METHODS AND MATERIALS FOR IDENTIFYING MALIGNANT SKIN LESIONS

Abstract

This document provides methods and materials for identifying malignant skin lesions (e.g., malignant pigmented skin lesions). For example, methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify malignant skin lesions are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 61/726,217, filed Nov. 14, 2012. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via electronic filing and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 7, 2013, is named sequence_listing.txt and is 101,321 bytes in size.

BACKGROUND

[0003] 1. Technical Field

[0004] This document relates to methods and materials for identifying malignant skin lesions (e.g., malignant pigmented skin lesions). For example, this document relates to methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify malignant skin lesions.

[0005] 2. Background Information

[0006] Malignant skin lesions are typically identified by obtaining a skin biopsy and morphologically assessing the biopsy's melanocytes under a microscope. Such a procedure can be difficult to standardize and can lead to overcalling of melanomas.

[0007] Once a diagnosis of melanoma is made by morphological assessment, the risk of metastasis is typically determined by the invasion depth of malignant cells into the skin (i.e., the Breslow depth). The Breslow depth can dictate further work-up such as a need for an invasive sentinel lymph node (SLN) procedure. Such procedures, however, can lead to inaccurate determinations of the true malignant potential of a pigmented lesion.

SUMMARY

[0008] This document provides methods and materials for identifying malignant skin lesions (e.g., malignant pigmented skin lesions). For example, this document provides methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify malignant skin lesions.

[0009] As described herein, quantitative PCR can be performed using a routine skin biopsy sample (e.g., a paraffin-embedded tissue biopsy) to obtain expression data (e.g., gene copy numbers) for one or more marker genes. Correction protocols can be used to reduce the impact of basal keratinocyte contamination on the analysis of the expression data from the test sample. For example, the contribution of gene expression from basal keratinocytes present within the test skin sample can be determined and removed from the overall gene expression values to determine the final gene expression value for a particular gene as expressed from cells other than basal keratinocytes (e.g., melanocytes). An assessment of the final gene expression values, which include minimal, if any, contribution from basal keratinocytes, for a collection of marker genes can be used to determine the benign or malignant biological behavior of the tested skin lesion.

[0010] In general, one aspect of this document features a method for identifying a malignant skin lesion. The method comprises, or consists essentially of, (a) determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, and CXCL1 to obtain a measured expression level of the marker gene for the test sample, (b) determining, within the test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of the keratinocyte marker gene for the test sample, (c) removing, from the measured expression level of the marker gene for the test sample, a level of expression attributable to keratinocytes present in the test sample using the measured expression level of the keratinocyte marker gene for the test sample and a keratinocyte correction factor to obtain a corrected value of marker gene expression for the test sample, and (d) identifying the test sample as containing a malignant skin lesion based, at least in part, on the corrected value of marker gene expression for the test sample. The keratinocyte marker gene can be K14. The marker gene can be SPP1. The step (c) can comprise (i) multiplying the measured expression level of the keratinocyte marker gene for the test sample by the keratinocyte correction factor to obtain a correction value and (ii) subtracting the correction value from the measured expression level of the marker gene for the test sample to obtain the corrected value of marker gene expression for the test sample. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0011] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0012] FIG. 1 is a flow chart of an exemplary process for determining the gene expression value, which includes minimal, if any, contribution from basal keratinocytes, for a marker gene by cells within a tested sample (e.g., a tested skin biopsy sample).

[0013] FIG. 2 is a flow chart of an exemplary process for determining a keratinocyte correction factor for a marker gene of interest.

[0014] FIG. 3 is a flow chart of an exemplary process for removing copy number contamination from basal keratinocytes from a copy number value for a marker gene to determine the gene expression value, which includes minimal, if any, contribution from basal keratinocytes, for that marker gene by cells within a tested sample (e.g., a tested skin biopsy sample).

[0015] FIG. 4 is a diagram of an example of a generic computer device and a generic mobile computer device that can be used as described herein.

[0016] FIG. 5 is a flow chart of an exemplary process for using FN1 and SPP1 expression levels to determine the benign or malignant nature of a skin lesion.

[0017] FIG. 6 is a flow chart of an exemplary process for using FN1 and ITGB3 expression levels to determine the benign or malignant nature of a skin lesion.

[0018] FIG. 7 is a network diagram.

DETAILED DESCRIPTION

[0019] This document provides methods and materials for identifying malignant skin lesions (e.g., malignant pigmented skin lesions). For example, this document provides methods and materials for using quantitative PCR results and correction protocols to reduce the impact of basal keratinocyte contamination on the analysis of test sample results to identify malignant skin lesions.

[0020] FIG. 1 shows an exemplary process 100 for determining a gene expression value, which includes minimal, if any, contribution from basal keratinocytes, for a marker gene by cells within a tested sample (e.g., a tested skin biopsy sample). The process begins at box 102, where quantitative PCR using a collection of primer sets and a test sample is used to obtain a Ct value for the target of each primer set. Each gene of interest can be assessed using a single primer set or multiple different primer sets (e.g., two, three, four, five, six, seven, or more different primer sets). In some cases, quantitative PCR is performed using each primer set and control nucleic acid of the target of each primer set (e.g., linearized cDNA fragments) to obtain a standard curve for each primer set as set forth in box 104. In some cases, quantitative PCR is performed using each primer set and a known sample as an internal control (e.g., a stock biological sample) to obtain an internal control value for each primer set as set forth in box 106. This internal control can be used to set values for each primer set across different assays. In some cases, the quantitative PCR performed according to boxes 102, 104, and 106 can be performed in parallel. For example, the quantitative PCR performed according to boxes 102, 104, and 106 can be performed in a single 96 well format.

[0021] At box 108, the quality of the obtained standard curves can be confirmed. In some cases, a gene of interest included in the assay format can be a melanocyte marker (e.g., levels of MLANA and/or MITF expression) to confirm the presence of melanocytes in the test sample. Other examples of melanocyte markers that can be used as described herein include, without limitation, TYR, TYRP1, DCT, PMEL, OCA2, MLPH, and MC1R.

[0022] At box 110, the raw copy number of each target present in the test sample is determined using the Ct values and the standard curve for each target. In some cases, the averaged, corrected copy number for each gene is calculated using the raw copy number of each target of a particular gene and the internal control value for each primer set (box 112). This averaged, corrected copy number value for each gene can be normalized to a set number of one or more housekeeping genes as set forth in box 114. For example, each averaged, corrected copy number value for each gene can be normalized to 100,000 copies of the combination of ACTB, RPL8, RPLP0, and B2M. Other examples of housekeeping genes that can be used as described herein include, without limitation, RRN18S, GAPD, PGK1, PPIA, RPL13A, YWHAZ, SDHA, TFRC, ALAS1, GUSB, HMBS, HPRT1, TBP, and TUPP. Once normalized, the copy number values for each gene can be referred to as the averaged, corrected, normalized copy number for that gene as present in the test sample.

[0023] At box 116, the averaged, corrected, normalized copy number for each gene can be adjusted to remove the copy number contamination from basal keratinocytes present in the test sample. In general, copy number contamination from basal keratinocytes can be removed by (a) determining a keratinocyte correction factor for the gene of interest using one or more keratinocyte markers (e.g., keratin 14 (K14)) and one or more normal skin samples (e.g., FFPE-embedded normal skin samples), (b) determining the averaged, corrected, normalized copy number value for the one or more keratinocyte markers of the test sample and multiplying that value by the keratinocyte correction factor to obtain a correction value for the gene of interest, and (c) subtracting that correction value from the averaged, corrected, normalized copy number value of the gene of interest to obtain the final copy number for the gene of interest. Examples of keratinocyte markers that can be used as described herein include, without limitation, KRTS, KRT1, KRT10, KRT17, ITGB4, ITGA6, PLEC, DST, and COL17A1.

[0024] With reference to FIG. 2, process 200 can be used to obtain a keratinocyte correction factor for a gene of interest. At box 202, the averaged, corrected, normalized copy number for one or more genes of interest (e.g., Gene X) and one or more basal keratinocyte marker genes (e.g., K14) are determined using one or more normal skin samples and procedures similar to those described in FIG. 1. As box 204, the keratinocyte correction factor for each gene of interest (e.g., Gene X) is determined by dividing the averaged, corrected, normalized copy number for each gene of interest present in a normal skin sample by the averaged, corrected, normalized copy number of a basal keratinocyte marker gene present in a normal skin sample. Examples of keratinocyte correction factors for particular genes of interest are set forth in Table E under column "AVG per copy K14."

[0025] With reference to FIG. 3, once a keratinocyte correction factor in determined for a particular gene of interest (e.g., Gene X), then the averaged, corrected, normalized copy number for the basal keratinocyte marker gene present in the test sample can be multiplied by the keratinocyte correction factor for the gene of interest (e.g., Gene X) to obtain a correction value for the gene of interest (e.g., Gene X). See, e.g., box 302. At box 304, the correction value for the gene of interest (e.g., Gene X) is subtracted from the averaged, corrected, normalized copy number for the gene of interest (e.g., Gene X) present in the test sample to obtain a final copy number value of the gene of interest (e.g., Gene X) present in the test sample.

[0026] FIG. 4 is a diagram of an example of a generic computer device 1400 and a generic mobile computer device 1450, which may be used with the techniques described herein. Computing device 1400 is intended to represent various forms of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other appropriate computers. Computing device 1450 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smart phones, and other similar computing devices. The components shown here, their connections and relationships, and their functions, are meant to be exemplary only, and are not meant to limit implementations of the inventions described and/or claimed in this document.

[0027] Computing device 1400 includes a processor 1402, memory 1404, a storage device 1406, a high-speed interface 1408 connecting to memory 1404 and high-speed expansion ports 1410, and a low speed interface 1415 connecting to low speed bus 1414 and storage device 1406. Each of the components 1402, 1404, 1406, 1408, 1410, and 1415, are interconnected using various busses, and may be mounted on a common motherboard or in other manners as appropriate. The processor 1402 can process instructions for execution within the computing device 1400, including instructions stored in the memory 1404 or on the storage device 1406 to display graphical information for a GUI on an external input/output device, such as display 1416 coupled to high speed interface 1408. In other implementations, multiple processors and/or multiple buses may be used, as appropriate, along with multiple memories and types of memory. Also, multiple computing devices 1400 may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multi-processor system).

[0028] The memory 1404 stores information within the computing device 1400. In one implementation, the memory 1404 is a volatile memory unit or units. In another implementation, the memory 1404 is a non-volatile memory unit or units. The memory 1404 may also be another form of computer-readable medium, such as a magnetic or optical disk.

[0029] The storage device 1406 is capable of providing mass storage for the computing device 1400. In one implementation, the storage device 1406 may be or contain a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disk device, or a tape device, a flash memory or other similar solid state memory device, or an array of devices, including devices in a storage area network or other configurations. A computer program product can be tangibly embodied in an information carrier. The computer program product may also contain instructions that, when executed, perform one or more methods, such as those described herein. The information carrier is a computer- or machine-readable medium, such as the memory 1404, the storage device 1406, memory on processor 1402, or a propagated signal.

[0030] The high speed controller 1408 manages bandwidth-intensive operations for the computing device 1400, while the low speed controller 1415 manages lower bandwidth-intensive operations. Such allocation of functions is exemplary only. In one implementation, the high-speed controller 1408 is coupled to memory 1404, display 1416 (e.g., through a graphics processor or accelerator), and to high-speed expansion ports 1410, which may accept various expansion cards (not shown). In the implementation, low-speed controller 1415 is coupled to storage device 1406 and low-speed expansion port 1414. The low-speed expansion port, which may include various communication ports (e.g., USB, Bluetooth, Ethernet, or wireless Ethernet) may be coupled to one or more input/output devices, such as a keyboard, a pointing device, a scanner, an optical reader, a fluorescent signal detector, or a networking device such as a switch or router, e.g., through a network adapter.

[0031] The computing device 1400 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a standard server 1420, or multiple times in a group of such servers. It may also be implemented as part of a rack server system 1424. In addition, it may be implemented in a personal computer such as a laptop computer 1422. In some cases, components from computing device 1400 may be combined with other components in a mobile device (not shown), such as device 1450. Each of such devices may contain one or more of computing device 1400, 1450, and an entire system may be made up of multiple computing devices 1400, 1450 communicating with each other.

[0032] Computing device 1450 includes a processor 1452, memory 1464, an input/output device such as a display 1454, a communication interface 1466, and a transceiver 1468, among other components (e.g., a scanner, an optical reader, a fluorescent signal detector). The device 1450 may also be provided with a storage device, such as a microdrive or other device, to provide additional storage. Each of the components 1450, 1452, 1464, 1454, 1466, and 1468, are interconnected using various buses, and several of the components may be mounted on a common motherboard or in other manners as appropriate.

[0033] The processor 1452 can execute instructions within the computing device 1450, including instructions stored in the memory 1464. The processor may be implemented as a chipset of chips that include separate and multiple analog and digital processors. The processor may provide, for example, for coordination of the other components of the device 1450, such as control of user interfaces, applications run by device 1450, and wireless communication by device 1450.

[0034] Processor 1452 may communicate with a user through control interface 1458 and display interface 1456 coupled to a display 1454. The display 1454 may be, for example, a TFT LCD (Thin-Film-Transistor Liquid Crystal Display) or an OLED (Organic Light Emitting Diode) display, or other appropriate display technology. The display interface 1456 may comprise appropriate circuitry for driving the display 1454 to present graphical and other information to a user. The control interface 1458 may receive commands from a user and convert them for submission to the processor 1452. In addition, an external interface 1462 may be provide in communication with processor 1452, so as to enable near area communication of device 1450 with other devices. External interface 1462 may provide, for example, for wired communication in some implementations, or for wireless communication in other implementations, and multiple interfaces may also be used.

[0035] The memory 1464 stores information within the computing device 1450. The memory 1464 can be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. Expansion memory 1474 may also be provided and connected to device 1450 through expansion interface 1472, which may include, for example, a SIMM (Single In Line Memory Module) card interface. Such expansion memory 1474 may provide extra storage space for device 1450, or may also store applications or other information for device 1450. For example, expansion memory 1474 may include instructions to carry out or supplement the processes described herein, and may include secure information also. Thus, for example, expansion memory 1474 may be provide as a security module for device 1450, and may be programmed with instructions that permit secure use of device 1450. In addition, secure applications may be provided via the SIMM cards, along with additional information, such as placing identifying information on the SIMM card in a non-hackable manner.

[0036] The memory may include, for example, flash memory and/or NVRAM memory, as discussed below. In one implementation, a computer program product is tangibly embodied in an information carrier. The computer program product contains instructions that, when executed, perform one or more methods, such as those described herein. The information carrier is a computer- or machine-readable medium, such as the memory 1464, expansion memory 1474, memory on processor 1452, or a propagated signal that may be received, for example, over transceiver 1468 or external interface 1462.

[0037] Device 1450 may communicate wirelessly through communication interface 1466, which may include digital signal processing circuitry where necessary. Communication interface 1466 may provide for communications under various modes or protocols, such as GSM voice calls, SMS, EMS, or MMS messaging, CDMA, TDMA, PDC, WCDMA, CDMA2000, or GPRS, among others. Such communication may occur, for example, through radio-frequency transceiver 1468. In addition, short-range communication may occur, such as using a Bluetooth, WiFi, or other such transceiver (not shown). In addition, GPS (Global Positioning System) receiver module 1470 may provide additional navigation- and location-related wireless data to device 1450, which may be used as appropriate by applications running on device 1450.

[0038] Device 1450 may also communicate audibly using audio codec 1460, which may receive spoken information from a user and convert it to usable digital information. Audio codec 1460 may likewise generate audible sound for a user, such as through a speaker, e.g., in a handset of device 1450. Such sound may include sound from voice telephone calls, may include recorded sound (e.g., voice messages, music files, etc.) and may also include sound generated by applications operating on device 1450.

[0039] The computing device 1450 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a cellular telephone 1480. It may also be implemented as part of a smartphone 1482, personal digital assistant, or other similar mobile device.

[0040] Various implementations of the systems and techniques described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and/or combinations thereof. These various implementations can include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0041] These computer programs (also known as programs, software, software applications or code) include machine instructions for a programmable processor, and can be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the terms "machine-readable medium" and "computer-readable medium" refer to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs)) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term "machine-readable signal" refers to any signal used to provide machine instructions and/or data to a programmable processor.

[0042] To provide for interaction with a user, the systems and techniques described herein can be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying information to the user and a keyboard and a pointing device (e.g., a mouse or a trackball) by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0043] The systems and techniques described herein can be implemented in a computing system that includes a back end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front end component (e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the systems and techniques described herein), or any combination of such back end, middleware, or front end components. The components of the system can be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include a local area network ("LAN"), a wide area network ("WAN"), and the Internet.

[0044] The computing system can include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0045] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Marker Genes that Discriminate Between Benign and Malignant Tissue

[0046] Marker genes were ordered by their ability to differentiate benign from malignant tissue (Table A). This was based on the analysis of 73 benign and 53 malignant tissues, and the hypothesis that changes in expression of fibronectin-associated gene networks are indicative of malignant cell behavior. Values of the test statistic were for the Wilcoxon rank sum test. The values of the test statistic for a Winsorized two-sample test (trimmed outliers were replaced with actual values) and for the chi-square test for the zero vs. >zero versions of each variable were included. The top 5 discriminatory genes based on each statistical test were highlighted in bold.

TABLE-US-00001 TABLE A Test statistic value Wilcoxon Winsorized rank sum two-sample Chi- gene test t-test square test FN1 -10.2312 -8.04081 106.714 SPP1 -9.0279 -4.9374 86.774 COL4A1 -8.8807 -7.27171 83.711 TNC -8.7511 -8.31049 75.549 ITGA3 -8.6008 -5.86334 79.788 LOXL3 -8.1978 -6.75327 75.144 AGRN -8.1243 -7.91238 62.611 VCAN -8.0812 -6.24088 67.388 PLOD3 -8.0384 -6.89248 62.691 ITGB1 -8.0021 -7.38143 59.973 PTK2 -7.5279 -7.19889 54.446 CTGF -7.4997 -5.581 57.79 PLOD1 -7.332 -7.36126 44.87 LAMC1 -7.2425 -6.1057 54.233 THBS1 -7.2425 -5.60331 54.233 LOXL2 -7.2241 -6.33208 55.909 IL6 -7.1777 -6.41883 56.966 LOXL1 -7.1279 -6.34431 52.878 IL8 -7.1194 -5.76042 57.296 CYR61 -6.741 -6.97388 43.866 ITGAV -6.5947 -6.27571 47.021 YAP -6.4848 -6.36431 42.417 BGN -6.3419 -6.01066 25.387 LAMB1 -6.3293 -5.68826 37.061 ITGB3 -6.3142 -5.13158 40.835 CXCL1 -6.1077 -5.66564 40.137 THBS2 -6.0427 -5.02003 37.413 COL18A1 -6.0379 -4.9125 41.339 SPARC -6.0272 -6.39324 38.098 TP53 -6.0182 -6.18554 34.945 PLOD2 -5.9082 -3.50272 47.576 CCL2 -5.8844 -5.38758 30.69 FBLN2 -5.5848 -4.59826 31.913 LAMA1 -5.4876 -4.2817 31.071 THBS4 -5.3971 -3.88786 35.27 COL1A1 -5.325 -4.37617 34.693 ITGA5 -4.9847 -3.56695 25.243 TAZ -4.036 -3.26011 18.313 POSTN -3.8054 -2.78378 19.813 LOX -3.728 -2.8677 17.157 CSRC -3.7078 -3.71759 13.983 LAMA3 -3.5805 -2.99652 13.391 CDKN1A -3.5766 -3.20447 17.228 CDKN2A -3.5491 -2.90903 15.938 ITGA2 -3.4083 -2.72495 11.766 LAMC2 -3.4083 -2.53784 11.766 PCOLCE2 -3.3469 -3.53676 14.449 LOXL4 -3.2079 -2.76128 10.943 PCOLCE -2.2172 -1.13805 7.993 LAMB3 -1.2822 0.89459 7.028 CSF2 2.175 1.93095 4.522

Example 2

Marker Panel Revision after Statistical Analysis

[0047] The candidate gene list from Example 1 was modified to include other FN1 network genes as well as four housekeeping genes (ACTB, RPLP0, RPL8, and B2M), two keratinocyte markers (K10 and K14) to assess keratinocyte contamination, and four melanocyte markers (MITF, TYR, MLANA and PMEL) to assess melanocyte content in the skin sections. Genes from Example 1 with low discriminatory value and a more distant neighborhood to FN1 were excluded from the test setup (LAMC1, LOXL2, CYR61, YAP, BGN, LAMB1, THBS2, COL18A1, SPARC, TP53, PLOD2, CCL2, FBLN2, LAMA1, THBS4, COL1A1, TAZ, POSTN, LOX, CSRC, LAMA3, CDKN1A, CDKN2A, LAMC2, PCOLCE2, LOXL4, PCOLCE, LAMB3, and CSF2). Instead, the discriminatory ability of other FN1 network genes was determined (PLAT, CSK, GDF15, FARP1, ARPC1B, NES, NTRK3, SNX17, L1CAM, and CD44). The following results were based on the analysis of 26 benign nevi and 52 primary cutaneous melanomas with documented subsequent metastasis or skin lesions of melanoma metastasis (Table B). The top 5 genes were highlighted.

TABLE-US-00002 TABLE B Test Statistic value Wilcoxon Winsorized rank sum two-sample Chi-square gene test t-test test COL4A1 -5.85975 -5.42545 46.3273 FN1 -5.50862 -3.63639 35.1951 PLAT -4.82670 -3.13568 25.7234 IL8 -4.61443 -4.41668 28.6000 SPP1 -4.60153 -3.08137 23.0816 PLOD3 -4.37001 -3.91553 18.8036 TNC -4.26431 -3.14128 19.5000 CXCL1 -4.24452 -3.76681 20.6471 CSK -4.15178 -2.96444 18.3962 GDF15 -4.01364 -2.99752 13.7083 ITGB3 -3.92608 -2.80068 16.3091 CCL2 -3.61870 -3.45423 17.5176 VCAN -3.46906 -2.26781 12.5593 ITGB1 -3.40897 -3.63399 5.0221 PLOD1 -3.40380 -3.20309 9.2625 CTGF -3.11725 -2.20507 10.0645 THBS1 -3.11721 -2.01257 10.0645 ITGA3 -3.04915 -2.65398 7.5341 FARP1 -2.99724 -2.28024 9.2857 AGRN -2.92104 -3.30679 1.8838 IL6 -2.85960 -3.05600 10.6257 LOXL3 -2.84999 -2.70498 5.1096 LOXL1 -2.69957 -2.11477 8.1250 ARPC1B -2.57571 -2.82320 All but 1 value >0 NES -2.45264 -2.70056 2.4375 PTK2 -2.22328 -2.26180 4.4057 ITGA2 -2.08353 -1.50078 4.4571 ITGA5 -1.93478 -1.39663 3.8451 ITGAV -1.29341 -0.81964 3.5615 NTRK3 -1.22485 75 of the 78 values are = 0 MITF 0.58305 0.73916 0.4274 SNX17 0.74754 0.90733 0.0785 L1CAM 1.61125 0.27151 2.1081 MLANA 2.96258 2.92548 All values >0 CD44 5.23089 7.17590 All but 1 value >0

[0048] Based on the results of Example 1 and above, FN1 was identified as a component of the melanoma phenotype that is at the core of a gene network that discriminates between benign and malignant melanocytic skin lesions (FIG. 7). The modeling was based on the STRING 9.0 database (string-db.org).

[0049] The list of all 71 genes tested is provided in Table 1.

TABLE-US-00003 TABLE 1 List of genes used to discriminate benign skin tissue lesions from malignant skin tissue lesions. GenBank .RTM. GenBank .RTM. Gene Name Accession No. GI No. FN1 NM_212482 47132556 NM_002026 47132558 NM_212474 47132548 NM_212476 47132552 NM_212478 47132554 NM_054034 47132546 SPP1 NM_001040058 91206461 NM_001040060 91598938 NM_000582 38146097 COL4A1 NM_001845 148536824 TNC NM_002160 340745336 ITGA3 NM_005501 171846264 NM_002204 171846266 LOXL3 NM_032603 22095373 AGRN NM_198576 344179122 VCAN NM_004385 255918074 NM_001164098 255918078 NM_001164097 255918076 PLOD3 NM_001084 62739167 ITGB1 NM_002211 182519230 NM_133376 182507162 NM_033668 182507160 PTK2 NM_001199649 313851043 NM_005607 313851042 NM_153831 313851041 CTGF NM_001901 98986335 PLOD1 NM_000302 324710986 LAMC1 NM_002293 145309325 THBS1 NM_003246 40317625 LOXL2 NM_002318 67782347 IL6 NM_000600 224831235 LOXL1 NM_005576 67782345 IL8 NM_000584 324073503 CYR61 NM_001554 197313774 ITGAV NM_001144999 223468594 NM_001145000 223468596 NM_002210 223468593 YAP NM_001130145 303523503 NM_001195045 303523626 NM_006106 303523510 NM_001195044 303523609 BGN NM_001711 268607602 LAMB1 NM_002291 167614503 ITGB3 NM_000212 47078291 CXCL1 NM_001511 373432598 THBS2 NM_003247 40317627 COL18A1 NM_030582 110611234 NM_130445 110611232 SPARC NM_003118 365777426 TP53 NM_000546 371502114 NM_001126112 371502115 NM_001126114 371502117 NM_001126113 371502116 PLOD2 NM_182943 62739164 NM_000935 62739165 CCL2 NM_002982 56119169 FBLN2 NM_001998 51873054 NM_001004019 51873052 NM_001165035 259013546 LAMA1 NM_005559 329112585 THBS4 NM_003248 291167798 COL1A1 NM_000088 110349771 ITGA5 NM_002205 56237028 TAZ NM_000116 195232764 NM_181311 195232766 NM_181312 195232765 NM_181313 195232767 POSTN NM_001135934 209862910 NM_006475 209862906 NM_001135935 209863010 LOX NM_001178102 296010939 NM_002317 296010938 CSRC NM_005417 38202215 NM_198291 38202216 LAMA3 NM_198129 38045909 NM_001127717 189217424 CDKN1A NM_000389 310832422 NM_001220777 334085239 NM_078467 310832423 NM_001220778 334085241 CDKN2A NM_000077 300863097 NM_058195 300863095 NM_001195132 304376271 ITGA2 NM_002203 116295257 LAMC2 NM_005562 157419137 NM_018891 157419139 PCOLCE2 NM_013363 296317252 LOXL4 NM_032211 67782348 PCOLCE NM_002593 157653328 LAMB3 NM_000228 62868214 NM_001017402 62868216 NM_001127641 189083718 CSF2 NM_000758 371502128 ACTB NM_001101 168480144 RPLP0 NM_053275 49087137 NM_001002 49087144 RPL8 NM_000973 72377361 NM_033301 15431305 B2M NM_004048 37704380 K10 NM_000421 195972865 K14 NM_000526 197313720 MITF NM_198158 296841082 NM_198177 296841080 NM_006722 296841079 NM_198159 296841078 NM_000248 296841081 NM_001184967 296841084 NM_198178 296923803 TYR NM_000372 113722118 MLANA NM_005511 5031912 PMEL NM_001200054 318037594 NM_001200053 318037592 NM_006928 318068057 NES NM_006617 38176299 L1CAM NM_024003 221316758 NM_001143963 221316759 NM_000425 221316755 GDF15 NM_004864 153792494 ARPC1B NM_005720 325197176 FARP1 NM_005766 48928036 NM_001001715 159032536 NTRK3 NM_001007156 340745351 NM_001012338 340745349 NM_001243101 340745352 NM_002530 340745350 CSK NM_001127190 187475372 NM_004383 187475371 CD44 NM_001001391 48255940 NM_001001392 48255942 NM_001202556 321400139 NM_001001389 48255936 NM_000610 48255934 NM_001001390 48255938 NM_001202555 321400137 NM_001202557 321400141 SNX17 NM_014748 388596703 PLAT NM_000930 132626665 NM_033011 132626641

[0050] Gene expression of target genes was assessed by SYBR/EVA-Green based RT-PCR. All tested genes were accompanied by a standard curve for quantification of absolute copy number per a defined number of housekeeping genes. mRNA extraction from paraffin-embedded biospecimen was performed using an extraction protocol (Qiagen RNA FFPE extraction kit) and an extraction robot (Qiacube from Qiagen). mRNA was transcribed into cDNA using a commercially available kit (iScript kit from BioRad), and Fluidigm technology was used for PCR cycling.

[0051] The primer design was performed using web-based open access software. The primers were HPLC purified to minimize background and were optimized for formalin-fixed, paraffin-embedded (FFPE) tissue (i.e., highly degraded tissue). The primers were designed to detect a maximum number of gene transcripts and were designed to be cDNA specific (i.e., not affected by genomic DNA contamination of the total, tissue-derived cDNA). The housekeeping genes, keratin genes, melanocyte-specific genes, and selected high interest genes were detected using four separate and individually designed primer pairs. The primer pairs are set forth in Table 2.

TABLE-US-00004 TABLE 2 Primer sets for indicated genes. Gene Name Forward primer Reverse primer ACTB 5'-GCCAACCGCGAGAAGATG-3'; 5'-GGCTGGGGTGTTGAAGGT-3'; SEQ ID NO: 1 SEQ ID NO: 2 5'-CGCGAGAAGATGACCCAGAT-3'; 5'-GGGGTGTTGAAGGTCTCAAA-3'; SEQ ID NO: 3 SEQ ID NO: 4 5'-TGACCCAGATCATGTTTGAGA-3'; 5'-GTACATGGCTGGGGTGTTG-3'; SEQ ID NO: 5 SEQ ID NO: 6 5'-CTGAACCCCAAGGCCAAC-3'; 5'-TGATCTGGGTCATCTTCTCG-3'; SEQ ID NO: 7 SEQ ID NO: 8 RPLP0 5'-AACTCTGCATTCTCGCTTCC-3'; 5'-GCAGACAGACACTGGCAACA-3'; SEQ ID NO: 9 SEQ ID NO: 10 5'-GCACCATTGAAATCCTGAGTG-3'; 5'-GCTCCCACTTTGTCTCCAGT-3'; SEQ ID NO: 11 SEQ ID NO: 12 5'-TCACAGAGGAAACTCTGCATTC-3'; 5'-GGACACCCTCCAGGAAGC-3'; SEQ ID NO: 13 SEQ ID NO: 14 5'-ATCTCCAGGGGCACCATT-3'; 5'-AGCTGCACATCACTCAGGATT-3'; SEQ ID NO: 15 SEQ ID NO: 16 RPL8 5'-ACTGCTGGCCACGAGTACG-3'; 5'-ATGCTCCACAGGATTCATGG-3'; SEQ ID NO: 17 SEQ ID NO: 18 5'-ACAGAGCTGTGGTTGGTGTG-3'; 5'-TTGTCAATTCGGCCACCT-3'; SEQ ID NO: 19 SEQ ID NO: 20 5'-TATCTCCTCAGCCAACAGAGC-3'; 5'-AGCCACCACACCAACCAC-3'; SEQ ID NO: 21 SEQ ID NO: 22 5'-GTGTGGCCATGAATCCTGT-3'; 5'-CCACCTCCAAAAGGATGCTC-3'; SEQ ID NO: 23 SEQ ID NO: 24 B2M 5'-TCTCTCTTTCTGGCCTGGAG-3'; 5'-GAATCTTTGGAGTACGCTGGA-3'; SEQ ID NO: 25 SEQ ID NO: 26 5'-TGGAGGCTATCCAGCGTACT-3'; 5'-CGTGAGTAAACCTGAATCTTTGG-3'; SEQ ID NO: 27 SEQ ID NO: 28 5'-CCAGCGTACTCCAAAGATTCA-3'; 5'-TCTCTGCTGGATGACGTGAG-3'; SEQ ID NO: 29 SEQ ID NO: 30 5'-GGCTATCCAGCGTACTCCAA-3'; 5'-GCTGGATGACGTGAGTAAACC-3'; SEQ ID NO: 31 SEQ ID NO: 32 KRT14 5'-ACCATTGAGGACCTGAGGAA-3'; 5'-GTCCACTGTGGCTGTGAGAA-3'; SEQ ID NO: 33 SEQ ID NO: 34 5'-CATTGAGGACCTGAGGAACA-3'; 5'-AATCTGCAGAAGGACATTGG-3'; SEQ ID NO: 35 SEQ ID NO: 36 5'-GATGACTTCCGCACCAAGTA-3'; 5'-CGCAGGTTCAACTCTGTCTC-3'; SEQ ID NO: 37 SEQ ID NO: 38 5'-TCCGCACCAAGTATGAGACA-3'; 5'-ACTCATGCGCAGGTTCAACT-3'; SEQ ID NO: 39 SEQ ID NO: 40 KRT10 5'-GAGCCTCGTGACTACAGCAA-3'; 5'-GCAGGATGTTGGCATTATCAGT-3'; SEQ ID NO: 41 SEQ ID NO: 42 5'-AAAACCATCGATGACCTTAAAAA-3'; 5'-GATCTGAAGCAGGATGTTGG-3'; SEQ ID NO: 43 SEQ ID NO: 44 MITF 5'-TTCCCAAGTCAAATGATCCAG-3'; 5'-AAGATGGTTCCCTTGTTCCA-3'; SEQ ID NO: 45 SEQ ID NO: 46 5'-CGGCATTTGTTGCTCAGAAT-3'; 5'-GAGCCTGCATTTCAAGTTCC-3'; SEQ ID NO: 47 SEQ ID NO: 48 TYR 5'-TTCCTTCTTCACCATGCATTT-3'; 5'-GGAGCCACTGCTCAAAAATA-3'; SEQ ID NO: 49 SEQ ID NO: 50 5'-TCCAAAGATCTGGGCTATGA-3'; 5'-TTGAAAAGAGTCTGGGTCTGAA-3'; SEQ ID NO: 51 SEQ ID NO: 52 MLANA 5'-GAGAAAAACTGTGAACCTGTGG-3'; 5'-ATAAGCAGGTGGAGCATTGG-3'; SEQ ID NO: 53 SEQ ID NO: 54 5'-GAAGACGAAATGGATACAGAGC-3'; 5'-GTGCCAACATGAAGACTTTTATC-3'; SEQ ID NO: 55 SEQ ID NO: 56 PMEL 5'-GTGGTCAGCACCCAGCTTAT-3'; 5'-CCAAGGCCTGCTTCTTGAC-3'; SEQ ID NO: 57 SEQ ID NO: 58 5'-GCTGTGGTCCTTGCATCTCT-3'; 5'-GCTTCATAAGTCTGCGCCTA-3'; SEQ ID NO: 59 SEQ ID NO: 60 FN1 5'-CTCCTGCACATGCTTTGGA-3'; 5'-AGGTCTGCGGCAGTTGTC-3'; SEQ ID NO: 61 SEQ ID NO: 62 5'-AGGCTTTGGAAGTGGTCATT-3'; 5'-CCATTGTCATGGCACCATCT-3'; SEQ ID NO: 63 SEQ ID NO: 64 5'-GAAGTGGTCATTTCAGATGTGATT-3'; 5'-CCATTGTCATGGCACCATCT-3'; SEQ ID NO: 65 SEQ ID NO: 66 5'-TGGTCATTTCAGATGTGATTCAT-3'; 5'-CATTGTCATGGCACCATCTA-3'; SEQ ID NO: 67 SEQ ID NO: 68 SPP1 5'-GTTTCGCAGACCTGACATCC-3'; 5'-TCCTCGTCTGTAGCATCAGG-3'; SEQ ID NO: 69 SEQ ID NO: 70 5'-CCTGACATCCAGTACCCTGA-3'; 5'-TGAGGTGATGTCCTCGTCTG-3'; SEQ ID NO: 71 SEQ ID NO: 72 5'-GAATCTCCTAGCCCCACAGA-3'; 5'-GGTTTCTTCAGAGGACACAGC-3'; SEQ ID NO: 73 SEQ ID NO: 74 5'-CCCATCTCAGAAGCAGAATCTC-3'; 5'-ACAGCATTCTGTGGGGCTA-3'; SEQ ID NO: 75 SEQ ID NO: 76 COL4A 1 5'-GGAAAACCAGGACCCAGAG-3'; 5'-CTTTTTCCCCTTTGTCACCA-3'; SEQ ID NO: 77 SEQ ID NO: 78 5'-AGAAAGGTGAACCCGGAAAA-3'; 5'-GGTTTGCCTCTGGGTCCT-3'; SEQ ID NO: 79 SEQ ID NO: 80 5'-GAGAAAAGGGCCAAAAAGGT-3'; 5'-CATCCCCTGAAATCCAGGTT-3'; SEQ ID NO: 81 SEQ ID NO: 82 5'-AAAGGGCCAAAAAGGTGAAC-3'; 5'-CCTGGCATCCCCTGAAAT-3'; SEQ ID NO: 83 SEQ ID NO: 84 TNC 5'-GTGTCAACCTGATGGGGAGA-3'; 5'-GTTAACGCCCTGACTGTGGT-3'; SEQ ID NO: 85 SEQ ID NO: 86 5'-GGTACAGTGGGACAGCAGGT-3'; 5'-GATCTGCCATTGTGGTAGGC-3'; SEQ ID NO: 87 SEQ ID NO: 88 5'-AACCACAGTCAGGGCGTTA-3'; 5'-GTTCGTGGCCCTTCCAGT-3'; SEQ ID NO: 89 SEQ ID NO: 90 5'-AAGCTGAAGGTGGAGGGGTA-3'; 5'-GAGTCACCTGCTGTCCCACT-3'; SEQ ID NO: 91 SEQ ID NO: 92 ITGA3 5'-TATTCCTCCGAACCAGCATC-3'; 5'-CACCAGCTCCGAGTCAATGT-3'; SEQ ID NO: 93 SEQ ID NO: 94 5'-CCACCATCAACATGGAGAAC-3'; 5'-AGTCAATGTCCACAGAGAACCA-3'; SEQ ID NO: 95 SEQ ID NO: 96 LOXL3 5'-CAACTGCCACATTGGTGATG-3'; 5'-AAACCTCCTGTTGGCCTCTT-3'; SEQ ID NO: 97 SEQ ID NO: 98 5'-TGACATCACGGATGTGAAGC-3'; 5'-GGGTTGATGACAACCTGGAG-3'; SEQ ID NO: 99 SEQ ID NO: 100 AGRN 5'-TGTGACCGAGAGCGAGAAG-3'; 5'-CAGGCTCAGTTCAAAGTGGTT-3'; SEQ ID NO: 101 SEQ ID NO: 102 5'-CGGACCTTTGTCGAGTACCT-3'; 5'-GTTGCTCTGCAGTGCCTTCT-3'; SEQ ID NO: 103 SEQ ID NO: 104 VCAN 5'-GACTTCCGTTGGACTGATGG-3'; 5'-TGGTTGGGTCTCCAATTCTC-3'; SEQ ID NO: 105 SEQ ID NO: 106 5'-ACGTGCAAGAAAGGAACAGT-3'; 5'-TCCAAAGGTCTTGGCATTTT-3'; SEQ ID NO: 107 SEQ ID NO: 108 PLOD3 5'-GCAGAGATGGAGCACTACGG-3'; 5'-CAGCCTTGAATCCTCATGC-3'; SEQ ID NO: 109 SEQ ID NO: 110 5'-GGAAGGAATCGTGGAGCAG-3'; 5'-CAGCAGTGGGAACCAGTACA-3'; SEQ ID NO: 111 SEQ ID NO: 112 ITGB1 5'-CTGATGAATGAAATGAGGAGGA-3'; 5'-CACAAATGAGCCAAATCCAA-3'; SEQ ID NO: 113 SEQ ID NO: 114 5'-CAGTTTGCTGTGTGTTTGCTC-3'; 5'-CATGATTTGGCATTTGCTTTT-3'; SEQ ID NO: 115 SEQ ID NO: 116 PTK2 5'-GCCCCACCAGAGGAGTATGT-3'; 5'-AAGCCGACTTCCTTCACCA-3'; SEQ ID NO: 117 SEQ ID NO: 118 5'-GAGACCATTCCCCTCCTACC-3'; 5'-GCTTCTGTGCCATCTCAATCT-3'; SEQ ID NO: 119 SEQ ID NO: 120 CTGF 5'-CGAAGCTGACCTGGAAGAGA-3'; 5'-TGGGAGTACGGATGCACTTT-3'; SEQ ID NO: 121 SEQ ID NO: 122 5'-GTGTGCACCGCCAAAGAT-3'; 5'-CGTACCACCGAAGATGCAG-3'; SEQ ID NO: 123 SEQ ID NO: 124 PLOD1 5'-CTACCCCGGCTACTACACCA-3'; 5'-GACAAAGGCCAGGTCAAACT-3'; SEQ ID NO: 125 SEQ ID NO: 126 5'-AGTCGGGGTGGATTACGAG-3'; 5'-ACAGTTGTAGCGCAGGAACC-3'; SEQ ID NO: 127 SEQ ID NO: 128 LAMC1 5'-ATGATGATGGCAGGGATGG-3'; 5'-GCATTGATCTCGGCTTCTTG-3'; SEQ ID NO: 129 SEQ ID NO: 130 THBS1 5'-CTGTGGCACACAGGAAACAC-3'; 5'-ACGAGGGTCATGCCACAG-3'; SEQ ID NO: 131 SEQ ID NO: 132 5'-GCCAAAGACGGGTTTCATTA-3'; 5'-GCCATGATTTTCTTCCCTTC-3'; SEQ ID NO: 133 SEQ ID NO: 134 LOXL2 5 '-CTCCTCCTACGGCAAGGGA-3'; 5'-TGGAGATTGTCTAACCAGATGGG-3'; SEQ ID NO: 135 SEQ ID NO: 136 5'-CTCCTACGGCAAGGGAGAAG-3'; 5'-TTGCCAGTACAGTGGAGATTG-3'; SEQ ID NO: 137 SEQ ID NO: 138 1L6 5'-CCAGAGCTGTGCAGATGAGT-3'; 5'-TGCATCTAGATTCTTTGCCTTTT-3'; SEQ ID NO: 139 SEQ ID NO: 140 LOXL1 5'-AGGGCACAGCAGACTTCCT-3'; 5'-TCGTCCATGCTGTGGTAATG-3'; SEQ ID NO: 141 SEQ ID NO: 142 5'-GCATGCACCTCTCATACCC-3'; 5'-CGCATTGTAGGTGTCATAGCA-3'; SEQ ID NO: 143 SEQ ID NO: 144 1L8 5'-CTTGGCAGCCTTCCTGATT-3'; 5'-GCAAAACTGCACCTTCACAC-3'; SEQ ID NO: 145 SEQ ID NO: 146 CYR61 5'-CGCTCTGAAGGGGATCTG-3'; 5'-ACAGGGTCTGCCCTCTGACT-3'; SEQ ID NO: 147 SEQ ID NO: 148 5'-GAGCTCAGTCAGAGGGCAGA-3'; 5'-AACTTTCCCCGTTTTGGTAGA-3'; SEQ ID NO: 149 SEQ ID NO: 150 ITGAV 5'-GACCTTGGAAACCCAATGAA-3'; 5'-TCCATCTCTGACTGCTGGTG-3'; SEQ ID NO: 431 SEQ ID NO: 432 5'-GGTGGTATGTGACCTTGGAAA-3'; 5'-GCACACTGAAACGAAGACCA-3'; SEQ ID NO: 439 SEQ ID NO: 440 YAP 5'-TGAACAGTGTGGATGAGATGG-3'; 5'-GCAGGGTGCTTTGGTTGATA-3'; SEQ ID NO: 151 SEQ ID NO: 152 BGN 5'-AAGGGTCTCCAGCACCTCTAC-3'; 5'-AAGGCCTTCTCATGGATCTT-3'; SEQ ID NO: 153 SEQ ID NO: 154 5'-GAGCTCCGCAAGGATGACT-3'; 5'-AGGACGAGGGCGTAGAGGT-3'; SEQ ID NO: 155 SEQ ID NO: 156 LAMB1 5'-CATTCAAGGAACCCAGAACC-3'; 5'-GCGTTGAACAAGGTTTCCTC-3'; SEQ ID NO: 157 SEQ ID NO: 158 ITGB3 5'-AAGAGCCAGAGTGTCCCAAG-3'; 5'-ACTGAGAGCAGGACCACCA-3'; SEQ ID NO: 159 SEQ ID NO: 160 5'-CTTCTCCTGTGTCCGCTACAA-3'; 5'-CATGGCCTGAGCACATCTC-3'; SEQ ID NO: 161 SEQ ID NO: 162 5'-TGCCTGCACCTTTAAGAAAGA-3'; 5'-CCGGTCAAACTTCTTACACTCC-3'; SEQ ID NO: 163 SEQ ID NO: 164 5'-AAGGGGGAGATGTGCTCAG-3'; 5'-CAGTCCCCACAGCTGCAC-3'; SEQ ID NO: 165 SEQ ID NO: 166 CXCL1 5'-AAACCGAAGTCATAGCCACAC-3'; 5'-AAGCTTTCCGCCCATTCTT-3'; SEQ ID NO: 167 SEQ ID NO: 168 THBS2 5'-AGGCCCAAGACTGGCTACAT-3'; 5'-CTGCCATGACCTGTTTTCCT-3'; SEQ ID NO: 169 SEQ ID NO: 170 5'-GGCAGGTGCGAACCTTATG-3'; 5'-CCTTCCAGCCAATGTTCCT-3'; SEQ ID NO: 171 SEQ ID NO: 172 COL18A1 5'-GATCGCTGAGCTGAAGGTG-3'; 5'-CGGATGCCCCATCTGAGT-3'; SEQ ID NO: 173 SEQ ID NO: 174 SPARC 5'-CCCATTGGCGAGTTTGAGAAG-3'; 5'-AGGAAGAGTCGAAGGTCTTGTT-3'; SEQ ID NO: 175 SEQ ID NO: 176 5'-GGAAGAAACTGTGGCAGAGG-3'; 5'-GGACAGGATTAGCTCCCACA-3'; SEQ ID NO: 177 SEQ ID NO: 178 TP53 5'-ACAACGTTCTGTCCCCCTTG-3'; 5'-GGGGACAGCATCAAATCATC-3'; SEQ ID NO: 179 SEQ ID NO: 180 PLOD2 5'-TGGATGCAGATGTTGTTTTGA-3'; 5'-CACAGCTTTCCATGACGAGTT-3'; SEQ ID NO: 181 SEQ ID NO: 182 5'-TTGATTGAACAAAACAGAAAGATCA-3'; 5'-TGACGAGTTACAAGAGGAGCAA-3'; SEQ ID NO: 183 SEQ ID NO: 184 CCL2 5'-CTGCTCATAGCAGCCACCTT-3'; 5'-AGGTGACTGGGGCATTGATT-3'; SEQ ID NO: 185 SEQ ID NO: 186 FBLN2 5'-ACGTGGAGGAGGACACAGAC-3'; 5'-GGAGCCTTCAGGGCTACTTC-3'; SEQ ID NO: 187 SEQ ID NO: 188 LAMA1 5'-AGCACTGCCAAAGTGGATG-3'; 5'-TTGTTGACATGGAACAAGACC-3'; SEQ ID NO: 189 SEQ ID NO: 190 THBS4 5'-GTGGGCTACATCAGGGTACG-3'; 5'-CAGAGTCAGCCACCAACTCA-3'; SEQ ID NO: 191 SEQ ID NO: 192 5'-CATCATCTGGTCCAACCTCA-3'; 5'-GTCCTCAGGGATGGTGTCAT-3'; SEQ ID NO: 193 SEQ ID NO: 194 COL1A1 5'-TGACCTCAAGATGTGCCACT-3'; 5'-TGGTTGGGGTCAATCCAGTA-3';

SEQ ID NO: 195 SEQ ID NO: 196 5'-GATGGATTCCAGTTCGAGTATG-3'; 5'-ATCAGGCGCAGGAAGGTC-3'; SEQ ID NO: 197 SEQ ID NO: 198 ITGA5 5'-CCCAAAAAGAGCGTCAGGT-3'; 5'-TTGTTGACATGGAACAAGACC-3'; SEQ ID NO: 199 SEQ ID NO: 200 TAZ 5'-CTTCCTAACAGTCCGCCCTA-3'; 5'-CCCGATCAGCACAGTGATTT-3'; SEQ ID NO: 201 SEQ ID NO: 202 POSTN 5'-CTGCTTCAGGGAGACACACC-3'; 5'-TGGCTTGCAACTTCCTCAC-3'; SEQ ID NO: 203 SEQ ID NO: 204 5'-AGGAAGTTGCAAGCCAACAA-3'; 5'-CGACCTTCCCTTAATCGTCTT-3'; SEQ ID NO: 205 SEQ ID NO: 206 LOX 5'-GCGGAGGAAAACTGTCTGG-3'; 5'-AAATCTGAGCAGCACCCTGT-3'; SEQ ID NO: 207 SEQ ID NO: 208 5'-ATATTCCTGGGAATGGCACA-3'; 5'-CCATACTGTGGTAATGTTGATGA-3'; SEQ ID NO: 209 SEQ ID NO: 210 CSRC 5'-TGTCAACAACACAGAGGGAGA-3'; 5'-CACGTAGTTGCTGGGGATGT-3'; SEQ ID NO: 211 SEQ ID NO: 212 5'-TGGCAAGATCACCAGACGG-3'; 5'-GGCACCTTTCGTGGTCTCAC-3'; SEQ ID NO: 213 SEQ ID NO: 214 LAMA3 5'-CATGTCGTCTTGGCTCACTC-3'; 5'-AAATTCTGGCCCCAACAATAC-3'; SEQ ID NO: 215 SEQ ID NO: 216 CDKN1A 5'-CATGTCGTCTTGGCTCACTC-3'; 5'-AAATTCTGGCCCCAACAATAC-3'; SEQ ID NO: 217 SEQ ID NO: 218 CDKN2A 5'-AGGAGCCAGCGTCTAGGG-3'; 5'-CTGCCCATCATCATGACCT-3'; SEQ ID NO: 219 SEQ ID NO: 220 5'-AACGCACCGAATAGTTACGG-3'; 5'-CATCATCATGACCTGGATCG-3'; SEQ ID NO: 221 SEQ ID NO: 222 ITGA2 5'-CACTGTTACGATTCCCCTGA-3'; 5'-CGGCTTTCTCATCAGGTTTC-3'; SEQ ID NO: 223 SEQ ID NO: 224 LAMC2 5'-ATTAGACGGCCTCCTGCATC-3'; 5'-AGACCAGCCCCTCTTCATCT-3'; SEQ ID NO: 225 SEQ ID NO: 226 PCOLCE2 5'-TACTTGGAAAATCACAGTTCCCG-3'; 5'-TGAATCGGAAATTGAGAACGACT-3'; SEQ ID NO: 443 SEQ ID NO: 444 LOXL4 5'-GGCCCCGGGAATTATATCT-3'; 5'-CCACTTCATAGTGGGGGTTC-3'; SEQ ID NO: 227 SEQ ID NO: 228 5'-CTGCACAACTGCCACACAG-3'; 5'-GTTCTGCATTGGCTGGGTAT-3'; SEQ ID NO: 229 SEQ ID NO: 230 PCOLCE 5'-CGTGGCAAGTGAGGGGTTC-3'; 5'-CGAAGACTCGGAATGAGAGGG-3'; SEQ ID NO: 231 SEQ ID NO: 232 5'-GAGGCTTCCTGCTCTGGT-3'; 5'-CGCAAAATTGGTGCTCAGT-3'; SEQ ID NO: 233 SEQ ID NO: 234 LAMB3 5'-GTCCGGGACTTCCTAACAGA-3'; 5'-GCTGACCTCCTGGATAGTGG-3'; SEQ ID NO: 235 SEQ ID NO: 236 PMEL 5'-GTGGTCAGCACCCAGCTTAT-3'; 5'-CCAAGGCCTGCTTCTTGAC-3'; SEQ ID NO: 237 SEQ ID NO: 238 5'-GCTGTGGTCCTTGCATCTCT-3'; 5'-GCTTCATAAGTCTGCGCCTA-3'; SEQ ID NO: 239 SEQ ID NO: 240 NES 5'-CTTCCCTCAGCTTTCAGGAC-3'; 5'-TCTGGGGTCCTAGGGAATTG-3'; SEQ ID NO: 241 SEQ ID NO: 242 5'-ACCTCAAGATGTCCCTCAGC-3'; 5'-CAGGAGGGTCCTGTACGTG-3'; SEQ ID NO: 243 SEQ ID NO: 244 LICAM 5'-GAGACCTTCGGCGAGTACAG-3'; 5'-AAAGGCCTTCTCCTCGTTGT-3'; SEQ ID NO: 245 SEQ ID NO: 246 5'-GGCGGCAAATACTCAGTGAA-3'; 5'-CCTGGGTGTCCTCCTTATCC-3'; SEQ ID NO: 247 SEQ ID NO: 248 GDF15 5'-CGGATACTCACGCCAGAAGT-3'; 5'-AGAGATACGCAGGTGCAGGT-3'; SEQ ID NO: 249 SEQ ID NO: 250 5'-AAGATTCGAACACCGACCTC-3'; 5'-GCACTTCTGGCGTGAGTATC-3'; SEQ ID NO: 251 SEQ ID NO: 252 ARPC1B 5'-CACGCCTGGAACAAGGAC-3'; 5'-ATGCACCTCATGGTTGTTGG-3'; SEQ ID NO: 253 SEQ ID NO: 254 5'-CAGGTGACAGGCATCGACT-3'; 5'-CGCAGGTCACAATACGGTTA-3'; SEQ ID NO: 255 SEQ ID NO: 256 FARP1 5'-TGAGGCCCTGAGAGAGAAGA-3'; 5'-ATTCCGAAACTCCACACGTC-3'; SEQ ID NO: 257 SEQ ID NO: 258 5'-TCAAGGAAATTGAGCAACGA-3'; 5'-TCTGATTTGGGCATTTGAGC-3'; SEQ ID NO: 259 SEQ ID NO: 260 NTRK3 5'-TATGGTCGACGGTCCAAAT-3'; 5'-TCCTCACCACTGATGACAGC-3'; SEQ ID NO: 261 SEQ ID NO: 262 5'-CACTGTGACCCACAAACCAG-3'; 5'-GCAAGTCCAACTGCTATGGA-3'; SEQ ID NO: 263 SEQ ID NO: 264 CSK 5'-TGAGGCCCTGAGAGAGAAGA-3'; 5'-ATTCCGAAACTCCACACGTC-3'; SEQ ID NO: 265 SEQ ID NO: 266 5'-TCTACTCCTTTGGGCGAGTG-3'; 5'-CGTCCTTCAGGGGAATTCTT-3'; SEQ ID NO: 267 SEQ ID NO: 268 CD44 5'-TAAGGACACCCCAAATTCCA-3'; 5'-GCCAAGATGATCAGCCATTC-3'; SEQ ID NO: 269 SEQ ID NO: 270 5'-GCAGTCAACAGTCGAAGAAGG-3'; 5'-AGCTTTTTCTTCTGCCCACA-3'; SEQ ID NO: 271 SEQ ID NO: 272 SNX17 5'-AGCCAGCAAGCAGTGAAGTC-3'; 5'-TCAGGTGACTCAAGCAGTGG-3'; SEQ ID NO: 273 SEQ ID NO: 274 5'-CCGGGAGTCTATGGTCAAAC-3'; 5'-CACGGCACTCAGCTTACTTG-3'; SEQ ID NO: 275 SEQ ID NO: 276 PLAT 5'-TGGAGCAGTCTTCGTTTCG-3'; 5'-CTGGCTCCTCTTCTGAATCG-3'; SEQ ID NO: 277 SEQ ID NO: 278 5'-GCCCGATTCAGAAGAGGAG-3'; 5'-TCATCTCTGCAGATCACTTGG-3'; SEQ ID NO: 279 SEQ ID NO: 280

[0052] The following was performed to generate a standard curve for the target of each primer pair. The standard was generated with a defined number of amplicons per volume for each primer pair. In particular, a standard (S7) was designed to contain about 5 million copies of amplicon-containing cDNA in a bacterial expression vector backbone (pJET1.2 obtained from Fermentas) per one microliter volume for each primer pair. From this, six 1:10 dilutions were generated such that seven standards S1 to S7 were obtained ranging from 5 to 5 million copies of amplicon. To obtain fragments of cDNA, total RNA was extracted from the human HaCaT, A431, and A375 cell lines, and the RNA was reverse transcribed into cDNA. Cell line-derived cDNA was used as a template to amplify fragments of cDNA that contained the desired amplicons for the real time-PCR primer pairs. A list of primers used to generate the desired cDNA fragments is listed in Table 3.

TABLE-US-00005 TABLE 3 Primer sets for generating cDNA fragments of the indicated genes. Gene Name Forward primer Reverse primer FN1 5'-CCAGCAGAGGCATAAGGTTC-3'; 5'-AGTAGTGCCTTCGGGACTGG-3'; SEQ ID NO: 281 SEQ ID NO: 282 SPP1 5'-AGGCTGATTCTGGAAGTTCTGAGG-3'; 5'-AATCTGGACTGCTTGTGGCTG-3'; SEQ ID NO: 283 SEQ ID NO: 284 COL4A1 5'-GTTGGGCCTCCAGGATTTA-3'; 5'-GCCTGGTAGTCCTGGGAAAC-3'; SEQ ID NO: 285 SEQ ID NO: 286 TNC 5'-TGGATGGATTGTGTTCCTGA-3'; 5'-GCCTGCCTTCAAGATTTCTG-3'; SEQ ID NO: 287 SEQ ID NO: 288 ITGA3 5'-CTGAGACTGTGCTGACCTGTG-3'; 5'-CTCTTCATCTCCGCCTTCTG-3'; SEQ ID NO: 289 SEQ ID NO: 290 LOXL3 5'-GAGACCGCCTACATCGAAGA-3'; 5'-GGTAGCGTTCAAACCTCCTG-3'; SEQ ID NO: 291 SEQ ID NO: 292 AGRN 5'-ACACCGTCCTCAACCTGAAG-3'; 5'-AATGGCCAGTGCCACATAGT-3'; SEQ ID NO: 293 SEQ ID NO: 294 VCAN 5'-GGTGCACTTTGTGAGCAAGA-3'; 5'-TTGGTATGCAGATGGGTTCA-3'; SEQ ID NO: 295 SEQ ID NO: 296 PLOD3 5'-AGCTGTGGTCCAACTTCTGG-3'; 5'-GTGTGGTAACCGGGAAACAG-3'; SEQ ID NO: 297 SEQ ID NO: 298 ITGB1 5'-TTCAGTTTGCTGTGTGTTTGC-3'; 5'-CCACCTTCTGGAGAATCCAA-3'; SEQ ID NO: 299 SEQ ID NO: 300 PTK2 5'-GGCAGTATTGACAGGGAGGA-3'; 5'-TACTCTTGCTGGAGGCTGGT-3'; SEQ ID NO: 301 SEQ ID NO: 302 CTGF 5'-GCCTATTCTGTCACTTCGGCTC-3'; 5'-GCAGGCACAGGTCTTGATGAAC-3'; SEQ ID NO: 303 SEQ ID NO: 304 PLOD1 5'-GACCTCTGGGAGGTGTTCAG-3'; 5'-TTAGGGATCGACGAAGGAGA-3'; SEQ ID NO: 305 SEQ ID NO: 306 LAMC1 5'-ATTCCTGCCATCAACCAGAC-3'; 5'-CCTGCTTCTTGGCTTCATTC-3'; SEQ ID NO: 307 SEQ ID NO: 308 THBS1 5'-CAAAGGGACATCCCAAAATG-3'; 5'-GAGTCAGCCATGATTTTCTTCC-3'; SEQ ID NO: 309 SEQ ID NO: 310 LOXL2 5'-TACCCCGAGTACTTCCAGCA-3'; 5'-GATCTGCTTCCAGGTCTTGC-3'; SEQ ID NO: 311 SEQ ID NO: 312 IL6 5'-CACACAGACAGCCACTCACC-3'; 5'-CAGGGGTGGTTATTGCATCT-3'; SEQ ID NO: 313 SEQ ID NO: 314 LOXL1 5'-CAGACCCCAACTATGTGCAA-3'; 5'-CGCATTGTAGGTGTCATAGCA-3'; SEQ ID NO: 315 SEQ ID NO: 316 IL8 5'-CTCTCTTGGCAGCCTTCCT-3'; 5'-TGAATTCTCAGCCCTCTTCAA-3'; SEQ ID NO: 317 SEQ ID NO: 318 CYR61 5'-TCGCCTTAGTCGTCACCCTT-3'; 5'-TGTTTCTCGTCAACTCCACCTCG-3'; SEQ ID NO: 319 SEQ ID NO: 320 ITGAV 5'-CTGATTTCATCGGGGTTGTC-3'; 5'-TGCCTTGCTGAATGAACTTG-3'; SEQ ID NO: 321 SEQ ID NO: 322 YAP 5'-CCAGTGAAACAGCCACCAC-3'; 5'-CTCCTTCCAGTGTTCCAAGG-3'; SEQ ID NO: 323 SEQ ID NO: 324 BGN 5'-GGACTCTGTCACACCCACCT-3'; 5'-CAGGGTCTCAGGGAGGTCTT-3'; SEQ ID NO: 325 SEQ ID NO: 326 LAMB1 5'-TGCCAGAGCTGAGATGTTGTT-3'; 5'-TGTAGCATTTCGGCTTTCCT-3'; SEQ ID NO: 327 SEQ ID NO: 328 ITGB3 5'-GGCAAGTACTGCGAGTGTGA-3'; 5'-ATTCTTTTCGGTCGTGGATG-3'; SEQ ID NO: 329 SEQ ID NO: 330 CXCL1 5'-CACTGCTGCTCCTGCTCCT-3'; 5'-TGTTCAGCATCTTTTCGATGA-3'; SEQ ID NO: 331 SEQ ID NO: 332 THBS2 5'-TGACAATGACAACATCCCAGA-3'; 5'-TGAGTCTGCCATGACCTGTT-3'; SEQ ID NO: 333 SEQ ID NO: 334 COL18A1 5'-CCCTGCTCTACACAGAACCAG-3'; 5'-ACACCTGGCTCCCCTTTCT-3'; SEQ ID NO: 335 SEQ ID NO: 336 SPARC 5'-GCCTGGATCTTCTTTCTCCTTTGC-3'; 5'-CATCCAGGGCGATGTACTTGTC-3'; SEQ ID NO: 337 SEQ ID NO: 338 TP53 5'-CCCCCTCTGAGTCAGGAAAC-3'; 5'-TCATGTGCTGTGACTGCTTG-3'; SEQ ID NO: 339 SEQ ID NO: 340 PLOD2 5'-TGGACCCACCAAGATTCTCCTG-3'; 5'-GACCACAGCTTTCCATGACGAG-3'; SEQ ID NO: 341 SEQ ID NO: 342 CCL2 5'-TCTGTGCCTGCTGCTCATAG-3'; 5'-GAGTTTGGGTTTGCTTGTCC-3'; SEQ ID NO: 343 SEQ ID NO: 344 FBLN2 5'-CGAGAAGTGCCCAGGAAG-3'; 5'-AGTGAGAAGCCAGGAAAGCA-3'; SEQ ID NO: 345 SEQ ID NO: 346 LAMA1 5'-TGGAAATATCACCCACAGCA-3'; 5'-AGGCATTTTTGCTTCACACC-3'; SEQ ID NO: 347 SEQ ID NO: 348 THBS4 5'-GCTCCAGCTTCTACGTGGTC-3'; 5'-TTAATTATCGAAGCGGTCGAA-3'; SEQ ID NO: 349 SEQ ID NO: 350 COL1A1 5'-AGCCAGCAGATCGAGAACAT-3'; 5'-CCTTCTTGAGGTTGCCAGTC-3'; SEQ ID NO: 351 SEQ ID NO: 352 ITGA5 5'-CACCAATCACCCCATTAACC-3'; 5'-GCTTGAGCTGAGCTTTTTCC-3'; SEQ ID NO: 353 SEQ ID NO: 354 TAZ 5'-CCAGGTGCTGGAAAAAGAAG-3'; 5'-GAGCTGCTCTGCCTGAGTCT-3'; SEQ ID NO: 355 SEQ ID NO: 356 POSTN 5'-GCAGACACACCTGTTGGAAA-3'; 5'-GAACGACCTTCCCTTAATCG-3'; SEQ ID NO: 357 SEQ ID NO: 358 LOX 5'-CCTACTACATCCAGGCGTCCAC-3'; 5'-ATGCAAATCGCCTGTGGTAGC-3'; SEQ ID NO: 359 SEQ ID NO: 360 CSRC 5'-CTGTTCGGAGGCTTCAACTC-3'; 5'-AGGGATCTCCCAGGCATC-3'; SEQ ID NO: 361 SEQ ID NO: 362 LAMAS 5'-TACCTGGGATCACCTCCATC-3'; 5'-ACAGGGATCCTCAGTGTCGT-3'; SEQ ID NO: 363 SEQ ID NO: 364 CDKN1A 5'-CGGGATGAGTTGGGAGGAG-3'; 5'-TTAGGGCTTCCTCTTGGAGA-3'; SEQ ID NO: 365 SEQ ID NO: 366 CDKN2A- 5'-ATGGTGCGCAGGTTCTTG-3'; 5'-ACCAGCGTGTCCAGGAAG-3'; 004 2A-201 SEQ ID NO: 367 SEQ ID NO: 368 CDKN2A- 5'-GAGCAGCATGGAGCCTTC-3'; 5'-GCATGGTTACTGCCTCTGGT-3'; 001 2A-202 SEQ ID NO: 369 SEQ ID NO: 370 ITGA2 5'-CAAACAGACAAGGCTGGTGA-3'; 5'-TCAATCTCATCTGGATTTTTGG-3'; SEQ ID NO: 371 SEQ ID NO: 372 LAMC2 5'-CTGCAGGTGGACAACAGAAA-3'; 5'-CATCAGCCAGAATCCCATCT-3'; SEQ ID NO: 373 SEQ ID NO: 374 PCOLCE2 5'-GTCCCCAGAGAGACCTGTTT-3'; 5'-AGACACAATTGGCGCAGGT-3'; SEQ ID NO: 375 SEQ ID NO: 376 LOXL4 5'-AAGACTGGACGCGATAGCTG-3'; 5'-GGTTGTTCCTGAGACGCTGT-3'; SEQ ID NO: 377 SEQ ID NO: 378 PCOLCE 5'-TACACCAGACCCGTGTTCCT-3'; 5'-TCCAGGTCAAACTTCTCGAAGG-3'; SEQ ID NO: 379 SEQ ID NO: 380 LAMBS 5'-CTTCAATGCCCAGCTCCA-3'; 5'-TTCCCAACCACATCTTCCAC-3'; SEQ ID NO: 381 SEQ ID NO: 382 CSF2 5'-CTGCTGCTCTTGGGCACT-3'; 5'-CAGCAGTCAAAGGGGATGAC-3'; SEQ ID NO: 383 SEQ ID NO: 384 ACTB 5'-AGGATTCCTATGTGGGCGACG-3'; 5'-TCAGGCAGCTCGTAGCTCTTC-3'; SEQ ID NO: 385 SEQ ID NO: 386 RPLP0 5'-GGAATGTGGGCTTTGTGTTCACC-3'; 5'-AGGCCAGGACTCGTTTGTACC-3'; SEQ ID NO: 387 SEQ ID NO: 388 RPL8 5'-ACATCAAGGGCATCGTCAAGG-3'; 5'-TCTCTTTCTCCTGCACAGTCTTGG-3'; SEQ ID NO: 389 SEQ ID NO: 390 B2M 5'-TGCTCGCGCTACTCTCTCTTTC-3'; 5'-TCACATGGTTCACACGGCAG-3'; SEQ ID NO: 391 SEQ ID NO: 392 K10 5'-TGGCCTTCTCTCTGGAAATG-3'; 5'-TCATTTCCTCCTCGTGGTTC-3'; SEQ ID NO: 393 SEQ ID NO: 394 K14 5'-AGGTGACCATGCAGAACCTC-3'; 5'-CCTCGTGGTTCTTCTTCAGG-3'; SEQ ID NO: 395 SEQ ID NO: 396 MITF 5'-GAAATCTTGGGCTTGATGGA-3'; 5'-CCGAGGTTGTTGTTGAAGGT-3'; SEQ ID NO: 397 SEQ ID NO: 398 TYR 5'-CCATGGATAAAGCTGCCAAT-3'; 5'-GACACAGCAAGCTCACAAGC-3'; SEQ ID NO: 399 SEQ ID NO: 400 MLANA 5'-CACTCTTACACCACGGCTGA-3'; 5'-CATAAGCAGGTGGAGCATTG-3'; SEQ ID NO: 401 SEQ ID NO: 402 PMEL 5'-TTGTCCAGGGTATTGAAAGTGC-3'; 5'-GACAAGAGCAGAAGATGCGGG-3'; SEQ ID NO: 403 SEQ ID NO: 404 NES 5'-GCGTTGGAACAGAGGTTGGAG-3'; 5'-CAGGTGTCTCAAGGGTAGCAGG-3'; SEQ ID NO: 405 SEQ ID NO: 406 L1CAM 5'-CTTCCCTTTCGCCACAGTATG-3'; 5'-CCTCCTTCTCCTTCTTGCCACT-3'; SEQ ID NO: 407 SEQ ID NO: 408 GDF15 5'-AATGGCTCTCAGATGCTCCTGG-3'; 5'-GATTCTGCCAGCAGTTGGTCC-3'; SEQ ID NO: 409 SEQ ID NO: 410 ARPC1B 5'-ACCACAGCTTCCTGGTGGAG-3'; 5'-GAGCGGATGGGCTTCTTGATG-3'; SEQ ID NO: 411 SEQ ID NO: 412 FARP1 5'-AACGTGACCTTGTCTCCCAAC-3'; 5'-GCATGACATCGCCGATTCTT-3'; SEQ ID NO: 413 SEQ ID NO: 414 NTRK3 5'-TTCAACAAGCCCACCCACTAC-3'; 5'-GTTCTCAATGACAGGGATGCG-3'; SEQ ID NO: 415 SEQ ID NO: 416 CSK 5'-CATGGAATACCTGGAGGGCAAC-3'; 5'-CAGGTGCCAGCAGTTCTTCAT-3'; SEQ ID NO: 417 SEQ ID NO: 418 CD44 5'-TCTCAGAGCTTCTCTACATCAC-3'; 5'-CTGACGACTCCTTGTTCACCA-3'; SEQ ID NO: 419 SEQ ID NO: 420 SNX17 5'-TCACCTCCTCTGTACCATTGC-3'; 5'-CTCATCTCCAATGCCCTCGA-3'; SEQ ID NO: 421 SEQ ID NO: 422 PLAT 5'-TGCAATGAAGAGAGGGCTCTG-3'; 5'-CGTGGCCCTGGTATCTATTTCA-3'; SEQ ID NO: 423 SEQ ID NO: 424

[0053] The PCR reactions were performed using a high-fidelity polymerase (product name: Phusion', obtained from New England Biolabs). PCR amplification products were checked for correct size and subsequently gel purified using the Qiagen Gel Extraction kit. Purified PCR fragments were subcloned into the bacterial expression vector pJET1.2 using a commercially available kit (Fermentas). The subcloned fragments were subsequently checked by restriction digest and DNA sequencing. Bacterial clones harboring the pJET1.2 expression vector with the correct PCR insert (containing the desired amplicon for real time PCR primer pairs) were frozen and stored at -80.degree. C. This was done to regenerate the same real time PCR standards over time.

[0054] Bacteria harboring the pJET1.2 expression vector with PCR inserts were cultured to generate sufficient amounts of vector. A small aliquot of the total retrieved expression vector with insert was linearized using the PvuI-HF restriction enzyme (from New England Biolabs). The digest was then purified using the Qiagen PCR purification kit. Linearized cDNA was diluted to a concentration of 20 ng/.mu.L. One .mu.L of each of a total of 71 linearized cDNA fragments (each at a 20 ng/.mu.L concentration) were mixed and brought to a final volume of 1 mL to obtain standard S7.

[0055] Standard S7 was then diluted six times at a 1:10 ratio to obtained standards S1 to S6. Dilution was performed using ultrapure water obtained from Promega (Cat. No. P1193).

[0056] The following was performed to generate cDNA from FFPE samples. FFPE blocks were cut at 20 .mu.m sections using a standard Leica microtome. For large pieces of tissue, 2.times.20 .mu.m full sections were used for RNA retrieval. For smaller tissues, up to 5.times.20 .mu.m sections were combined for RNA retrieval. RNA extraction was performed using the Qiagen RNA FFPE retrieval kit and a Qiagen QiaCube extraction robot. 0.5 to 1 .mu.g of RNA with a 260/280 ratio of greater than 1.8 were transcribed into cDNA using the BioRad iScript cDNA Synthesis kit. All biospecimens were annotated with clinical data from Mayo Clinic databases. H&E stained sections were obtained for each block analyzed and digitalized using a high-resolution slide scanner.

[0057] Fluidigm RT-PCR was performed using a 96.times.96 format for high throughput analysis (i.e., 96 cDNAs were analyzed for 96 markers; 9216 data points). The primer pairs and cDNAs were prepared in a 96 well format. Standard curves were calculated for each primer pair. Copy numbers per 100,000 housekeeping genes were calculated for each primer pair and averaged per gene. This was initially done for cDNAs derived from FFPE-embedded skin. To correct for epidermal cell-derived cross-contamination, background signal per one copy of K14 (a basal keratinocyte marker) was calculated from FFPE-embedded normal skin samples for each primer pair and averaged. Experimental samples were then normalized first to 100,000 housekeeping genes and then background-corrected for epidermal cross-contamination based on K14 copy number. In particular, the keratinocyte correction factor used for each gene is set forth in Table E under the column titled "AVG per copy K14."

[0058] The study design (Example 1) involved a comparison of the expression profile of `true` benign pigmented skin lesions (nevi, n=73) with `true` malignant melanomas of the skin. The latter comprised i) primary skin melanomas that were documented to metastasize, either to regional lymph nodes, to other areas of skin (in-transit), or to other organs; and ii) in-transit or comparison of nevi to in-transit melanoma metastases (n=54).

[0059] Tables C and D summarize the comparisons of the gene expressions between the 73 benign and 54 metastatic. Table A compares the ranked values using the Wilcoxon rank sum test, and Table E compares the dichotomized values (zero vs. >0) using the chi-square test.

[0060] A recursive partitioning approach was used to identify cut-points for the genes that would discriminate between these two groups. After partitioning the data at a cut-point of 45 for FN1, no further additional splits in the data based on the other genes were identified by this method.

[0061] Using a cutoff of 45 for FN1, the sensitivity was 92.6%, and the specificity was 98.6%. These results are provided in Tables 4 and 5 along with the next possible cutoff for FN1 at 124

TABLE-US-00006 TABLE 4 Frequency Percent Row Pct Col Pct Malignant Benign Total FN1 4 72 76 <45 3.15 56.69 59.84 5.26 94.74 7.41 98.63 FN1 50 1 51 >=45 39.37 0.79 40.16 98.04 1.96 92.59 1.37 Total 54 73 127 42.52 57.48 100.00

TABLE-US-00007 TABLE 5 Frequency Percent Row Pct Col Pct Malignant Benign Total FN1 8 73 81 <124 6.30 57.48 63.78 9.88 90.12 14.81 100.00 FN1 >=124 46 0 46 36.22 0.00 36.22 100.00 0.00 85.19 0.00 Total 54 73 127 42.52 57.48 100.00

[0062] The ability to further discriminate between the groups was assessed by considering SPP1 or ITGB3 in addition to FN1.

Benign Vs. Malignant--Option 1 Using FN1 and SPP1 (FIG. 5)

[0063] The results are set forth in Table 6.

TABLE-US-00008 TABLE 6 RULE for FIG. 5 Malignant Benign FN1 <45 and SPP1 = 0 2 72 FN1 >=45 52 1 or (FN1 <45 and SPP1 >0) Total 54 73

Benign Vs. Malignant--Option 2 Using FN1 and ITGB3 (FIG. 6)

[0064] The results are set forth in Table 7.

TABLE-US-00009 TABLE 7 RULE for FIG. 6 Malignant Benign FN1 <45 and ITGB3 = 0 3 72 FN1 >=45 51 1 or (FN1 <45 and ITGB3 >0) Total 54 73

[0065] If all three genes are included, the rule was as follows:

[0066] FN1<45 and SPP1=0 and ITGB3=0 denotes a negative test

[0067] vs.

[0068] all other combinations denotes a positive test.

[0069] This rule resulted in a specificity of 72/73 (98.6%), and a sensitivity of 53/54 (98.2%) (Table 8). Compared to a rule using FN1 alone, the specificity stayed the same but the sensitivity increased from 92.6% to 98.2% using this new rule.

TABLE-US-00010 TABLE 8 FN1 SPP1 ITGB3 malignant Frequency <45 Zero Zero No 72 <45 Zero Zero Yes 1 False Neg ID MM150 (case added from the Breslow file) >=45 Zero Zero No 1 False Pos ID N29 >=45 Zero Zero Yes 9 >=45 Zero >0 Yes 1 >=45 >0 Zero Yes 18 >=45 >0 >0 Yes 22 <45 Zero >0 Yes 1 <45 >0 Zero Yes 2

[0070] The rule was evaluated using 25 additional malignant patients who did not have mets (from the "Breslow" file). For 19 of these 25 patients, the rule was `negative` (Table 9).

TABLE-US-00011 TABLE 9 FN1 SPP1 ITGB3 Frequency <45 Zero Zero 19 <45 >0 Zero 1 >=45 Zero Zero 2 >=45 >0 Zero 3 <45 1

[0071] The rule also was evaluated using 33 thin melanomas (Table 10). For 25 of these 33 patients, the rule was `negative`.

TABLE-US-00012 TABLE 10 FN1 SPP1 ITGB3 Frequency <45 Zero Zero 25 <45 Zero >0 1 >=45 Zero Zero 5 >=45 >0 Zero 2

TABLE-US-00013 TABLE C Comparison of gene expression between benign and malignant Benign (N = 73) Malignant (N = 54) p value CXCL1_AVG_NORM <0.0001 N 73 54 Mean (SD) 4.8 (18.4) 20.0 (26.1) Median 0.0 10.3 Q1, Q3 0.0, 0.0 0.3, 31.1 Range (0.0-141.7) (0.0-120.4) CSF2_AVG_NORM 0.0482 N 73 54 Mean (SD) 10.5 (44.1) 4.3 (8.4) Median 2.5 1.0 Q1, Q3 0.6, 7.0 0.0, 4.0 Range (0.0-375.0) (0.0-41.0) CCL2_AVG_NORM <0.0001 N 73 54 Mean (SD) 37.0 (99.4) 244.2 (360.9) Median 0.0 112.8 Q1, Q3 0.0, 9.1 7.2, 342.2 Range (0.0-572.0) (0.0-1777.1) IL8_AVG_NORM <0.0001 N 73 54 Mean (SD) 125.5 (671.3) 53.2 (160.8) Median 0.0 13.0 Q1, Q3 0.0, 0.0 2.1, 52.5 Range (0.0-5058.7) (0.0-1171.7) IL6_AVG_NORM <0.0001 N 73 54 Mean (SD) 9.9 (69.1) 21.6 (35.0) Median 0.0 8.8 Q1, Q3 0.0, 0.0 0.3, 25.2 Range (0.0-589.1) (0.0-152.3) ITGA5_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 9.8 (26.8) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 7.0 Range (0.0-0.0) (0.0-168.0) ITGA3_AVG_NORM <0.0001 N 73 54 Mean (SD) 3.2 (27.5) 168.2 (313.4) Median 0.0 50.2 Q1, Q3 0.0, 0.0 2.0, 160.5 Range (0.0-235.4) (0.0-1506.0) ITGA2_AVG_NORM 0.0007 N 73 54 Mean (SD) 0.0 (0.0) 2.6 (10.0) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 0.0 Range (0.0-0.0) (0.0-69.7) ITGAV_AVG_NORM <0.0001 N 73 54 Mean (SD) 3.3 (23.9) 22.0 (32.9) Median 0.0 8.0 Q1, Q3 0.0, 0.0 0.0, 31.0 Range (0.0-199.9) (0.0-176.8) ITGB3_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 43.6 (90.3) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 52.5 Range (0.0-0.0) (0.0-495.3) ITGB1_AVG_NORM <0.0001 N 73 54 Mean (SD) 29.9 (95.1) 616.2 (742.2) Median 0.0 400.2 Q1, Q3 0.0, 0.0 84.7, 869.0 Range (0.0-487.9) (0.0-3877.9) FN1_AVG_NORM <0.0001 N 73 54 Mean (SD) 2.9 (15.6) 1570.9 (1949.8) Median 0.0 898.4 Q1, Q3 0.0, 0.0 299.5, 2186.1 Range (0.0-123.2) (0.0-11073.5) THBS1_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 85.1 (136.1) Median 0.0 16.8 Q1, Q3 0.0, 0.0 0.0, 153.8 Range (0.0-0.0) (0.0-786.2) THBS2_AVG_NORM <0.0001 N 73 54 Mean (SD) 25.9 (113.4) 280.0 (513.5) Median 0.0 44.1 Q1, Q3 0.0, 0.0 0.0, 340.1 Range (0.0-729.2) (0.0-3030.5) THBS4_AVG_NORM <0.0001 N 73 54 Mean (SD) 38.5 (151.2) 228.2 (663.7) Median 0.0 22.5 Q1, Q3 0.0, 0.0 0.0, 97.9 Range (0.0-1130.3) (0.0-3977.7) VCAN_AVG_NORM <0.0001 N 73 54 Mean (SD) 3.0 (21.7) 202.4 (262.8) Median 0.0 103.4 Q1, Q3 0.0, 0.0 0.0, 283.5 Range (0.0-181.3) (0.0-1113.2) BGAN_AVG_NORM <0.0001 N 73 54 Mean (SD) 69.3 (121.0) 422.4 (573.1) Median 0.0 248.5 Q1, Q3 0.0, 97.9 113.5, 462.9 Range (0.0-496.3) (0.0-3348.1) SPP1_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 1490.2 (3397.4) Median 0.0 338.1 Q1, Q3 0.0, 0.0 4.9, 1577.7 Range (0.0-0.0) (0.0-22427.0) TNC_AVG_NORM <0.0001 N 73 54 Mean (SD) 66.4 (240.1) 800.1 (808.7) Median 0.0 495.8 Q1, Q3 0.0, 0.0 174.5, 1322.9 Range (0.0-1393.3) (0.0-3162.2) SPARC_AVG_NORM <0.0001 N 73 54 Mean (SD) 843.7 (2222.8) 3208.4 (3182.6) Median 0.0 2895.8 Q1, Q3 0.0, 0.0 407.2, 5216.3 Range (0.0-11175.6) (0.0-13631.9) AGRN_AVG_NORM <0.0001 N 73 54 Mean (SD) 4.7 (18.1) 51.2 (53.8) Median 0.0 42.1 Q1, Q3 0.0, 0.0 10.7, 69.7 Range (0.0-121.7) (0.0-242.0) CTGF_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.4 (3.6) 90.9 (231.6) Median 0.0 22.1 Q1, Q3 0.0, 0.0 0.0, 125.9 Range (0.0-30.6) (0.0-1631.4) CYR61_AVG_NORM <0.0001 N 73 54 Mean (SD) 4.8 (13.0) 27.2 (39.2) Median 0.0 18.7 Q1, Q3 0.0, 0.0 4.9, 32.2 Range (0.0-70.4) (0.0-267.2) LAMA3_AVG_NORM 0.0004 N 73 54 Mean (SD) 1.1 (9.0) 1.2 (2.9) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 0.0 Range (0.0-76.8) (0.0-11.3) LAMC1_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 70.6 (159.4) Median 0.0 28.4 Q1, Q3 0.0, 0.0 0.0, 99.3 Range (0.0-0.0) (0.0-1136.2) LAMB1_AVG_NORM <0.0001 N 73 54 Mean (SD) 9.2 (38.4) 221.1 (354.3) Median 0.0 73.1 Q1, Q3 0.0, 0.0 0.0, 339.8 Range (0.0-248.8) (0.0-1877.6) LAMA1_AVG_NORM <0.0001 N 73 54 Mean (SD) 5.7 (14.5) 65.4 (149.0) Median 0.0 10.6 Q1, Q3 0.0, 0.0 0.0, 49.0 Range (0.0-76.5) (0.0-754.3) LAMC2_AVG_NORM 0.0003 N 73 54 Mean (SD) 0.0 (0.0) 4.0 (15.3) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 0.0 Range (0.0-0.0) (0.0-91.1) LAMB3_AVG_NORM 0.1473 N 73 54 Mean (SD) 33.5 (60.3) 32.2 (54.5) Median 0.0 12.1 Q1, Q3 0.0, 44.6 0.0, 37.0 Range (0.0-323.9) (0.0-246.0) COL1A1_AVG_NORM <0.0001 N 73 54 Mean (SD) 1534.4 (4365.3) 4191.6 (5865.9) Median 0.0 1704.4 Q1, Q3 0.0, 0.0 0.0, 6850.9 Range (0.0-22510.2) (0.0-31867.0) COL4A1_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.0 (0.0) 211.8 (344.1) Median 0.0 118.4 Q1, Q3 0.0, 0.0 2.3, 261.2 Range (0.0-0.0) (0.0-1774.4) COL18A1_AVG_NORM <0.0001 N 73 54 Mean (SD) 94.2 (783.4) 22.8 (38.8) Median 0.0 4.1 Q1, Q3 0.0, 0.0 0.0, 34.4 Range (0.0-6695.7) (0.0-208.8) LOX_AVG_NORM 0.0003 N 73 54 Mean (SD) 37.7 (132.8) 65.0 (113.9) Median 0.0 3.5 Q1, Q3 0.0, 0.0 0.0, 58.0 Range (0.0-991.2) (0.0-443.3) LOXL1_AVG_NORM <0.0001 N 73 54 Mean (SD) 0.8 (7.1) 39.6 (60.3) Median 0.0 18.5 Q1, Q3 0.0, 0.0 0.0, 65.0 Range (0.0-60.4) (0.0-349.0) LOXL2_AVG_NORM <0.0001 N 73 54 Mean (SD) 43.3 (356.8) 68.5 (129.9) Median 0.0 22.1 Q1, Q3 0.0, 0.0 0.0, 89.1 Range (0.0-3048.4) (0.0-821.4) LOXL3_AVG_NORM <0.0001 N 73 54 Mean (SD) 2.2 (12.3) 28.4 (71.1) Median 0.0 9.2 Q1, Q3 0.0, 0.0 2.5, 29.4 Range (0.0-89.7) (0.0-507.5) LOXL4_AVG_NORM 0.0010 N 73 54 Mean (SD) 33.8 (91.0) 129.1 (300.4) Median 0.0 9.1 Q1, Q3 0.0, 10.2 0.0, 67.0 Range (0.0-529.2) (0.0-1230.0) PLOD1_AVG_NORM <0.0001 N 73 54 Mean (SD) 33.7 (116.5) 420.3 (532.2) Median 0.0 242.3 Q1, Q3 0.0, 0.0 90.2, 659.3 Range (0.0-878.2) (0.0-3336.8) PLOD2_AVG_NORM <0.0001 N 73 54 Mean (SD) 44.5 (151.7) 314.8 (1284.4) Median 0.0 53.7 Q1, Q3 0.0, 0.0 2.3, 103.3 Range (0.0-1124.0) (0.0-9110.5) PLOD3_AVG_NORM <0.0001 N 73 54 Mean (SD) 2.7 (11.9) 68.0 (81.2) Median 0.0 38.3 Q1, Q3 0.0, 0.0 4.2, 101.9 Range (0.0-87.4) (0.0-330.2) PCOLCE2_AVG_NORM 0.0010 N 73 54 Mean (SD) 7.7 (25.8) 6.4 (14.9) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 3.1

Range (0.0-104.8) (0.0-68.4) PCOLCE_AVG_NORM 0.0232 N 73 54 Mean (SD) 92.1 (159.7) 170.4 (339.4) Median 0.0 40.9 Q1, Q3 0.0, 122.2 0.0, 175.1 Range (0.0-699.2) (0.0-1945.2) PTK2_AVG_NORM <0.0001 N 73 54 Mean (SD) 2.8 (14.4) 76.6 (81.8) Median 0.0 70.0 Q1, Q3 0.0, 0.0 0.0, 127.7 Range (0.0-116.5) (0.0-323.3) CSRC_AVG_NORM 0.0001 N 73 54 Mean (SD) 19.0 (40.9) 45.1 (65.9) Median 0.3 19.6 Q1, Q3 0.0, 24.8 4.2, 46.6 Range (0.0-266.6) (0.0-290.2) CDKN1A_AVG_NORM 0.0005 N 73 54 Mean (SD) 78.5 (150.9) 181.0 (271.7) Median 0.0 84.2 Q1, Q3 0.0, 118.9 0.0, 253.3 Range (0.0-788.2) (0.0-1083.2) CDKN2A_AVG_NORM 0.0002 N 73 54 Mean (SD) 6.1 (19.6) 9.7 (25.8) Median 0.0 1.0 Q1, Q3 0.0, 0.0 0.0, 6.9 Range (0.0-113.2) (0.0-175.1) TP53_AVG_NORM <0.0001 N 73 54 Mean (SD) 40.6 (98.6) 231.2 (289.8) Median 0.0 166.9 Q1, Q3 0.0, 0.0 0.0, 359.9 Range (0.0-410.8) (0.0-1722.4) YAP_AVG_NORM <0.0001 N 73 54 Mean (SD) 7.8 (36.6) 112.4 (161.4) Median 0.0 63.1 Q1, Q3 0.0, 0.0 0.0, 173.5 Range (0.0-246.3) (0.0-769.0) TAZ_AVG_NORM <0.0001 N 73 54 Mean (SD) 12.2 (27.9) 32.8 (44.3) Median 0.0 15.0 Q1, Q3 0.0, 0.7 0.0, 49.0 Range (0.0-122.7) (0.0-186.4) MITF_AVG_NORM <0.0001 N 73 54 Mean (SD) 251.0 (399.5) 569.8 (494.8) Median 45.5 467.3 Q1, Q3 0.0, 331.5 184.9, 777.8 Range (0.0-2143.3) (0.0-2200.0) MLANA_AVG_NORM 0.1823 N 73 54 Mean (SD) 3596.0 (3671.3) 4865.4 (4966.1) Median 2446.8 2803.5 Q1, Q3 950.9, 5019.4 1210.7, 6773.0 Range (14.0-17180.3) (62.8-19672.1) TYR_AVG_NORM 0.0040 N 73 54 Mean (SD) 349.7 (301.8) 839.8 (996.3) Median 254.3 515.1 Q1, Q3 119.5, 527.5 161.0, 1244.9 Range (0.0-1169.8) (2.0-5500.0) POSTN_AVG_NORM 0.0001 N 73 54 Mean (SD) 1138.7 (2155.7) 1933.9 (2318.1) Median 191.6 1252.0 Q1, Q3 0.0, 1449.9 397.4, 2457.4 Range (0.0-11078.1) (0.0-11193.2) FBLN2_AVG_NORM <0.0001 N 73 54 Mean (SD) 2.1 (17.3) 26.5 (42.2) Median 0.0 0.0 Q1, Q3 0.0, 0.0 0.0, 48.8 Range (0.0-148.2) (0.0-150.9)

TABLE-US-00014 TABLE D Comparison of gene expression between benign and malignant Benign Malignant (N = 73) (N = 54) p value CXCL1_AVG_NORM01 <0.0001 Zero 58 (79.5%) 12 (22.2%) >0 15 (20.5%) 42 (77.8%) CSF2_AVG_NORM01 0.0398 Zero 15 (20.5%) 20 (37.0%) >0 58 (79.5%) 34 (63.0%) CCL2_AVG_NORM01 <0.0001 Zero 53 (72.6%) 12 (22.2%) >0 20 (27.4%) 42 (77.8%) IL8_AVG_NORM01 <0.0001 Zero 63 (86.3%) 10 (18.5%) >0 10 (13.7%) 44 (81.5%) IL6_AVG_NORM01 <0.0001 Zero 65 (89.0%) 13 (24.1%) >0 8 (11.0%) 41 (75.9%) ITGA5_AVG_NORM01 <0.0001 Zero 73 (100.0%) 38 (70.4%) >0 0 (0.0%) 16 (29.6%) ITGA3_AVG_NORM01 <0.0001 Zero 72 (98.6%) 13 (24.1%) >0 1 (1.4%) 41 (75.9%) ITGA2_AVG_NORM01 0.0007 Zero 73 (100.0%) 46 (85.2%) >0 0 (0.0%) 8 (14.8%) ITGAV_AVG_NORM01 <0.0001 Zero 71 (97.3%) 24 (44.4%) >0 2 (2.7%) 30 (55.6%) ITGB3_AVG_NORM01 <0.0001 Zero 73 (100.0%) 30 (55.6%) >0 0 (0.0%) 24 (44.4%) ITGB1_AVG_NORM01 <0.0001 Zero 64 (87.7%) 11 (20.4%) >0 9 (12.3%) 43 (79.6%) FN1_AVG_NORM01 <0.0001 Zero 69 (94.5%) 2 (3.7%) >0 4 (5.5%) 52 (96.3%) THBS1_AVG_NORM01 <0.0001 Zero 73 (100.0%) 24 (44.4%) >0 0 (0.0%) 30 (55.6%) THBS2_AVG_NORM01 <0.0001 Zero 67 (91.8%) 23 (42.6%) >0 6 (8.2%) 31 (57.4%) THBS4_AVG_NORM01 <0.0001 Zero 58 (79.5%) 15 (27.8%) >0 15 (20.5%) 39 (72.2%) VCAN_AVG_NORM01 <0.0001 Zero 71 (97.3%) 16 (29.6%) >0 2 (2.7%) 38 (70.4%) BGAN_AVG_NORM01 <0.0001 Zero 42 (57.5%) 7 (13.0%) >0 31 (42.5%) 47 (87.0%) SPP1_AVG_NORM01 <0.0001 Zero 73 (100.0%) 12 (22.2%) >0 0 (0.0%) 42 (77.8%) TNC_AVG_NORM01 <0.0001 Zero 60 (82.2%) 3 (5.6%) >0 13 (17.8%) 51 (94.4%) SPARC_AVG_NORM01 <0.0001 Zero 57 (78.1%) 13 (24.1%) >0 16 (21.9%) 41 (75.9%) AGRN_AVG_NORM01 <0.0001 Zero 59 (80.8%) 5 (9.3%) >0 14 (19.2%) 49 (90.7%) CTGF_AVG_NORM01 <0.0001 Zero 72 (98.6%) 21 (38.9%) >0 1 (1.4%) 33 (61.1%) CYR61_AVG_NORM01 <0.0001 Zero 56 (76.7%) 9 (16.7%) >0 17 (23.3%) 45 (83.3%) LAMA3_AVG_NORM01 0.0003 Zero 72 (98.6%) 43 (79.6%) >0 1 (1.4%) 11 (20.4%) LAMC1_AVG_NORM01 <0.0001 Zero 73 (100.0%) 24 (44.4%) >0 0 (0.0%) 30 (55.6%) LAMB1_AVG_NORM01 <0.0001 Zero 66 (90.4%) 22 (40.7%) >0 7 (9.6%) 32 (59.3%) LAMA1_AVG_NORM01 <0.0001 Zero 57 (78.1%) 16 (29.6%) >0 16 (21.9%) 38 (70.4%) LAMC2_AVG_NORM01 0.0003 Zero 73 (100.0%) 45 (83.3%) >0 0 (0.0%) 9 (16.7%) LAMB3_AVG_NORM01 0.0061 Zero 45 (61.6%) 20 (37.0%) >0 28 (38.4%) 34 (63.0%) COL1A1_AVG_NORM01 <0.0001 Zero 60 (82.2%) 17 (31.5%) >0 13 (17.8%) 37 (68.5%) COL4A1_AVG_NORM01 <0.0001 Zero 73 (100.0%) 13 (24.1%) >0 0 (0.0%) 41 (75.9%) COL18A1_AVG_NORM01 <0.0001 Zero 64 (87.7%) 18 (33.3%) >0 9 (12.3%) 36 (66.7%) LOX_AVG_NORM01 <0.0001 Zero 60 (82.2%) 26 (48.1%) >0 13 (17.8%) 28 (51.9%) LOXL1_AVG_NORM01 <0.0001 Zero 72 (98.6%) 23 (42.6%) >0 1 (1.4%) 31 (57.4%) LOXL2_AVG_NORM01 <0.0001 Zero 70 (95.9%) 19 (35.2%) >0 3 (4.1%) 35 (64.8%) LOXL3_AVG_NORM01 <0.0001 Zero 69 (94.5%) 10 (18.5%) >0 4 (5.5%) 44 (81.5%) LOXL4_AVG_NORM01 0.0006 Zero 53 (72.6%) 23 (42.6%) >0 20 (27.4%) 31 (57.4%) PLOD1_AVG_NORM01 <0.0001 Zero 59 (80.8%) 12 (22.2%) >0 14 (19.2%) 42 (77.8%) PLOD2_AVG_NORM01 <0.0001 Zero 59 (80.8%) 10 (18.5%) >0 14 (19.2%) 44 (81.5%) PLOD3_AVG_NORM01 <0.0001 Zero 66 (90.4%) 11 (20.4%) >0 7 (9.6%) 43 (79.6%) PCOLCE2_AVG_NORM01 0.0002 Zero 66 (90.4%) 34 (63.0%) >0 7 (9.6%) 20 (37.0%) PCOLCE_AVG_NORM01 0.0036 Zero 42 (57.5%) 17 (31.5%) >0 31 (42.5%) 37 (68.5%) PTK2_AVG_NORM01 <0.0001 Zero 67 (91.8%) 16 (29.6%) >0 6 (8.2%) 38 (70.4%) CSRC_AVG_NORM01 0.0001 Zero 36 (49.3%) 9 (16.7%) >0 37 (50.7%) 45 (83.3%) CDKN1A_AVG_NORM01 0.0001 Zero 48 (65.8%) 16 (29.6%) >0 25 (34.2%) 38 (70.4%) CDKN2A_AVG_NORM01 <0.0001 Zero 57 (78.1%) 23 (42.6%) >0 16 (21.9%) 31 (57.4%) TP53_AVG_NORM01 <0.0001 Zero 59 (80.8%) 16 (29.6%) >0 14 (19.2%) 38 (70.4%) YAP_AVG_NORM01 <0.0001 Zero 68 (93.2%) 22 (40.7%) >0 5 (6.8%) 32 (59.3%) TAZ_AVG_NORM01 <0.0001 Zero 54 (74.0%) 19 (35.2%) >0 19 (26.0%) 35 (64.8%) MITF_AVG_NORM01 <0.0001 Zero 26 (35.6%) 2 (3.7%) >0 47 (64.4%) 52 (96.3%) MLANA_AVG_NORM01 >0 73 (100.0%) 54 (100.0%) TYR_AVG_NORM01 0.2202 Zero 2 (2.7%) 0 (0.0%) >0 71 (97.3%) 54 (100.0%) POSTN_AVG_NORM01 <0.0001 Zero 32 (43.8%) 4 (7.4%) >0 41 (56.2%) 50 (92.6%) FBLN2_AVG_NORM01 <0.0001 Zero 71 (97.3%) 31 (57.4%) >0 2 (2.7%) 23 (42.6%)

TABLE-US-00015 TABLE E MM79_CN MM80_CN MM81_CN MM82_CN AVG AVG AVG AVG AVG per copy per copy per copy per copy per copy K14 K14 K14 K14 K14 STDEV % STDE KRT14_AVG_NORM 1 1 1 1 1 0.000 KRT10_AVG_NORM 2.209 2.229 2.92 3.015 2.593 0.434 17 MITF_AVG_NORM 0.021 0.018 0.016 0.015 0.018 0.003 15 MLANA_AVG_NORM 0.021 0.018 0.016 0.015 0.018 0.003 15 TYR_AVG_NORM 0.004 0.002 0.002 0.001 0.002 0.001 56 PMEL_AVG_NORM 0.025 0.027 0.03 0.018 0.025 0.005 20% FN1_AVG_NORM 0.077 0.065 0.035 0.042 0.055 0.020 36 SPARC_AVG_NORM 1.294 1.143 0.568 1.707 1.178 0.471 40 AGRN_AVG_NORM 0.004 0.006 0.003 0.002 0.004 0.002 46 THBS1_AVG_NORM 0.064 0.015 0.018 0.005 0.026 0.026 103 THBS2_AVG_NORM 0.366 0.061 0.104 0.057 0.147 0.148 100 THBS4_AVG_NORM 0.018 0.006 0.005 0.001 0.008 0.007 98 VCAN_AVG_NORM 0.095 0.034 0.04 0.027 0.049 0.031 64 BGAN_AVG_NORM 0.015 0.027 0.014 0.015 0.018 0.006 35 COL1A1_AVG_NORM 1.695 3.44 0.689 6.695 3.130 2.635 84 COL4A1_AVG_NORM 0.069 0.026 0.03 0.016 0.035 0.023 66 COL4A2_AVG_NORM 0.115 0.042 0.041 0.004 0.051 0.046 92 COL18A1_AVG_NORM 0.015 0.009 0.005 0.002 0.008 0.006 73 CTGF_AVG_NORM 0.012 0.008 0.016 0.004 0.010 0.005 52 LOX_AVG_NORM 0.029 0.021 0.028 0.021 0.025 0.004 18 LOXL1_AVG_NORM 0.015 0.009 0.016 0.015 0.014 0.003 23 LOXL2_AVG_NORM 0.016 0.011 0.008 0.006 0.010 0.004 42 LOXL3_AVG_NORM 0.003 0.002 0.002 0.001 0.002 0.001 41 LOXL4_AVG_NORM 0.02 0.004 0.003 0.001 0.007 0.009 125 PLOD2_AVG_NORM 0.018 0.014 0.007 0.001 0.010 0.008 75 PLOD1_AVG_NORM 0.069 0.053 0.026 0.017 0.041 0.024 58 SPP1_AVG_NORM 0.092 0.002 0.007 0 0.025 0.045 177 TNC_AVG_NORM 0.025 0.02 0.027 0.013 0.021 0.006 29 PCOLCE2_AVG_NORM 0.011 0.001 0.006 0 0.005 0.005 113 PCOLCE_AVG_NORM 0.028 0.049 0.032 0.04 0.037 0.009 25 PLOD3_AVG_NORM 0.03 0.006 0.007 0.002 0.011 0.013 113 ITGB3_AVG_NORM 0.03 0.006 0.007 0.002 0.011 0.013 113 ITGB1_AVG_NORM 0.164 0.054 0.074 0.038 0.083 0.056 68 FBLN2_AVG_NORM 0.049 0.022 0.02 0.016 0.027 0.015 56 CYR61_AVG_NORM 0.006 0.002 0.003 0 0.003 0.003 91 ITGA5_AVG_NORM 0.011 0.005 0.007 0.003 0.007 0.003 53 ITGA3_AVG_NORM 0.016 0.008 0.006 0.008 0.010 0.004 47 ITGA2_AVG_NORM 0.08 0.034 0.019 0.084 0.054 0.033 60 ITGAV_AVG_NORM 0.013 0.005 0.003 0.003 0.006 0.005 79 CSRC_AVG_NORM 0.006 0.003 0.005 0.001 0.004 0.002 59 PTK2_AVG_NORM 0.035 0.02 0.011 0.009 0.019 0.012 63 POSTN_AVG_NORM 0.077 0.092 0.117 0.193 0.120 0.052 43 YAP_AVG_NORM 0.079 0.029 0.033 0.031 0.043 0.024 56 CXCL1_AVG_NORM 0.002 0 0 0 0.001 0.001 200 CSF2_AVG_NORM 0.002 0 0 0 0.001 0.001 200 CCL2_AVG_NORM 0.039 0.018 0.013 0.008 0.020 0.014 70 IL8_AVG_NORM 0.003 0 0.001 0 0.001 0.001 141 IL6_AVG_NORM 0.001 0 0 0 0.000 0.001 200 LAMA3_AVG_NORM 0.038 0.012 0.021 0.011 0.021 0.013 61 TP53_AVG_NORM 0.08 0.04 0.039 0.052 0.053 0.019 36 CDKN1A_AVG_NORM 0.057 0.029 0.037 0.014 0.034 0.018 52 CDKN2A_AVG_NORM 0.003 0.001 0.001 0 0.001 0.001 101 TAZ_AVG_NORM 0.026 0.008 0.008 0.003 0.011 0.010 90 LAMC1_AVG_NORM 0.062 0.013 0.016 0.008 0.025 0.025 101 LAMB1_AVG_NORM 0.046 0.019 0.026 0.008 0.025 0.016 65 LAMA1_AVG_NORM 0.007 0 0.001 0 0.002 0.003 168 LAMC2_AVG_NORM 0.034 0.009 0.012 0.016 0.018 0.011 63 LAMB3_AVG_NORM 0.042 0.016 0.026 0.017 0.025 0.012 48 PLAT_AVG_NORM 0.032 0.02 0.034 0.04 0.032 0.001 27 CSK_AVG_NORM 0.027 0.034 0.021 0.041 0.031 0.001 28 GDF15_AVG_NORM 0.029 0.019 0.033 0.019 0.025 0.001 28 FARP1_AVG_NORM 0.019 0.029 0.022 0.031 0.025 0.001 22 ARPC1B_AVG_NORM 0.015 0.03 0.042 0.018 0.026 0.012 47 NES_AVG_NORM 0.114 0.125 0.112 0.084 0.109 0.017 16 NTRK3_AVG_NORM 0.021 0.025 0.022 0.033 0.025 0.001 25 SNX17_AVG_NORM 0.112 0.099 0.089 0.123 0.106 0.015 14 L1CAM_AVG_NORM 0.017 0.04 0.01 0.024 0.023 0.013 56 CD44_AVG_NORM 0.112 0.089 0.09 0.123 0.104 0.017 16 indicates data missing or illegible when filed

[0072] The results provided herein demonstrate the development of a method for determining absolute levels (copy numbers) of genes of interest (e.g., FN-associated genes) from paraffin-embedded tissue by generating a highly defined internal standard that can be regenerated indefinitely. This standardization approach can allow for the comparison of results from independent experiments and thus, allows for extensive validation. The RT-PCR not only produced strong signals from highly degraded RNA due to FFPE embedding, but also was amendable to high-throughput analysis and was highly cost effective. While the methods provided herein were validated for melanoma, these methods are likely applicable to other human cancers. The results provided herein also demonstrate the discrimination between benign and malignant pigmented lesions based on multiple markers.

Example 3

Additional Marker Panel

[0073] A test kit panel was designed to include primers for assessing expression levels of eight marker genes (ITGB3, TNC, SPP1, SPARC, PLAT, COL4A1, PLOD3, and PTK2) as well as three housekeeping genes (ACTB, RPLP0, and RPL8), one keratinocyte markers (K14) to assess keratinocyte contamination, and two melanocyte markers (MLANA and MITF) to assess melanocyte content in the skin sections. The primers designed for this collection are set forth in Table 11.

TABLE-US-00016 TABLE 11 Primers for marker panel kit. SEQ Primer pair ID Gene name Direction Sequence NO: ACTB ACTB-G -F TGCTATCCCTGTACGCCTCT 433 ACTB-G -R GAGTCCATCACGATGCCAGT 434 ACTB ACTB-H -F GGACTTCGAGCAAGAGATGG 435 ACTB-H -R CTTCTCCAGGGAGGAGCTG 436 ACTB ACTB-I -F GGCTACAGCTTCACCACCAC 425 ACTB-I -R TAATGTCACGCACGATTTCC 426 RPLP0 RPLP0-B -F AACTCTGCATTCTCGCTTCC 9 RPLP0-B -R GCAGACAGACACTGGCAACA 10 RPLP0 RPLP0-C -F GCACCATTGAAATCCTGAGTG 11 RPLP0-C -R GCTCCCACTTTGTCTCCAGT 12 RPL8 RPL8-B -F ACAGAGCTGTGGTTGGTGTG 19 RPL8-B -R TTGTCAATTCGGCCACCT 20 RPL8 RPL8-E -F ACTGCTGGCCACGAGTACG 17 RPL8-E -R ATGCTCCACAGGATTCATGG 18 KRT14 KRT14-D -F TCCGCACCAAGTATGAGACA 39 KRT14-D -R ACTCATGCGCAGGTTCAACT 40 KRT14 KRT14-F -F GATGCAGATTGAGAGCCTGA 437 KRT14-F -R TTCTTCAGGTAGGCCAGCTC 438 MLANA MLANA-C -F GAGAAAAACTGTGAACCTGTGG 53 MLANA-C -R ATAAGCAGGTGGAGCATTGG 54 MITF MITF-B -F CGGCATTTGTTGCTCAGAAT 47 MITF-B -R GAGCCTGCATTTCAAGTTCC 48 ITGB3 ITGB3-A -F AAGAGCCAGAGTGTCCCAAG 159 ITGB3-A -R ACTGAGAGCAGGACCACCA 160 ITGB3 ITGB3-B -F CTTCTCCTGTGTCCGCTACAA 161 ITGB3-B -R CATGGCCTGAGCACATCTC 162 PLAT PLAT-C -F CCCAGCCAGGAAATCCAT 427 PLAT-C -R CTGGCTCCTCTTCTGAATCG 428 PLAT PLAT-D -F CAGTGCCTGTCAAAAGTTGC 429 PLAT-D -R CCCCGTTGAAACACCTTG 430 PLAT PLAT-E -F GAAGGATTTGCTGGGAAGTG 441 PLAT-E -R CGTGGCCCTGGTATCTATTT 442 PLOD3 PLOD3-D -F GGAAGGAATCGTGGAGCAG 111 PLOD3-D -R CAGCAGTGGGAACCAGTACA 112 PTK2 PTK2-D -F GAGACCATTCCCCTCCTACC 119 PTK2-D -R GCTTCTGTGCCATCTCAATCT 120 CDKN2A CDKN2A1-C -F AGGAGCCAGCGTCTAGGG 219 CDKN2A1-C -R CTGCCCATCATCATGACCT 220 CDKN2A CDKN2A2-C -F AACGCACCGAATAGTTACGG 221 CDKN2A2-C -R CATCATCATGACCTGGATCG 222

[0074] One purpose of the kit was to differentiate between melanoma with high and low risk of regional metastasis, and to appropriately select patients for surgical procedures such as sentinel lymph node biopsy (SLNB) or total lymphadenectomy. Another purpose of this kit was to estimate disease-free survival, disease relapse, or likelihood of death from melanoma. To study the ability of these methods to discriminate between melanoma with high and low risk of metastasis and to establish superiority to established methods, a cohort of 158 patients between October 1998 and June 2013 were identified as having been diagnosed with high-risk melanoma and as having underwent SLNB with the intention to assess metastatic potential of the tumor. Of note, high-risk melanoma by current criteria are defined as melanoma with an invasion depth (Breslow depth) of .gtoreq.1 mm; or melanoma with an invasion depth of 0.75 to 0.99 mm plus the presence of either one of the following three risk factors: >0 mitotic figures/mm.sup.2; tumor ulceration present; patient age <40 years.

[0075] All 158 patients met the criteria for high risk. 136 patients had a Breslow Depth .gtoreq.1 mm. 22 patients had a Breslow Depth between 0.75 and 0.99 and had at least 1 of the aforementioned 3 risk factors (ulceration, mitotic rate >0, age <40). Of the 158 patients, 36 (22.8%) had a melanoma-positive SLNB.

[0076] To select genes for a test kit from a pool of genes, the expression level of 52 genes (variables) was initially determined and dichotomized as zero vs. >zero and evaluated for an association with positive SLNB using the chi-square test for a 2.times.2 contingency level. The genes are ordered based on the value of the chi-square test statistic (Table 12).

TABLE-US-00017 TABLE 12 Value of the chi-square test statistic variable ITGB3 68.3522 SPP1 25.8460 LOXL3 16.7683 PLAT 16.5721 LAMB1 15.7544 YAP 13.4049 PLOD3 12.6062 TP53 12.3662 COL4A1 11.8336 TNC 11.3862 IL8 10.4697 ITGA5 10.3561 COL1A1 10.0006 VCAN 9.3250 PLOD1 8.6959 FN1 8.4857 PTK2 7.9874 ITGAV 7.7181 LOXL1 7.2109 LOXL2 6.6348 ITGB1 6.3556 CDKN1A 6.3117 CTGF 6.2588 GDF15 5.96939 CSRC 5.4435 ITGA2 5.0326 ITGA3 4.0603 LOX 3.8697 COL18A1 3.3392 IL6 3.0435 DSPP 2.7822 NTRK3 2.7822 LOXL4 2.7279 THBS2 2.5110 SPARC 1.9884 PCOLCE2 1.6499 AGRN 1.6118 CXCL1 1.3483 TAZ 1.3458 THBS4 1.1281 PCOLCE 0.9198 FBLN2 0.9198 LAMC2 0.9157 CCL2 0.8701 CDKN2A 0.6047 CSF2 0.5408 CYR61 0.4713 BGAN 0.4364 LAMA3 0.3455 POSTN 0.1902 LAMB3 0.1058 PLOD2 0.0152

[0077] As can be deduced from the chi-square test statistic, ITGB3 was highly discriminatory between melanoma with and without regional lymph node metastasis. The n (%) with a positive SLNB for those with no expression vs. expression level >0 was summarized (Table 13).

TABLE-US-00018 TABLE 13 Overall Positive No. (% of 158) No. (% of each row) FN1_01 Zero 110 (69.6%) 18 (16.4%) >0 48 (30.4%) 18 (37.5%) SPP1_01 Zero 93 (58.9%) 8 (8.6%) >0 65 (41.1%) 28 (43.1%) ITGB3_01 Zero 107 (67.7%) 4 (3.7%) >0 51 (32.3%) 32 (62.7%) TNC_01 Zero 114 (72.2%) 18 (15.8%) >0 44 (27.8%) 18 (40.9%) PLAT_01 Missing 18 0 Zero 83 (59.3%) 11 (13.3%) >0 57 (40.7%) 25 (43.9%) COL4A1_01 Zero 111 (70.3%) 17 (15.3%) >0 47 (29.7%) 19 (40.4%) SPARC_01 Missing 4 0 Zero 138 (89.6%) 30 (21.7%) >0 16 (10.4%) 6 (37.5%) AGRN_01 Missing 4 0 Zero 23 (14.9%) 3 (13.0%) >0 131 (85.1%) 33 (25.2%) THBS1_01 Missing 135 33 Zero 18 (78.3%) 0 (0.0%) >0 5 (21.7%) 3 (60.0%) THBS2_01 Missing 4 0 Zero 114 (74.0%) 23 (20.2%) >0 40 (26.0%) 13 (32.5%) THBS4_01 Missing 4 0 Zero 136 (88.3%) 30 (22.1%) >0 18 (11.7%) 6 (33.3%) VCAN_01 Missing 4 0 Zero 137 (89.0%) 27 (19.7%) >0 17 (11.0%) 9 (52.9%) BGAN_01 Missing 4 0 Zero 97 (63.0%) 21 (21.6%) >0 57 (37.0%) 15 (26.3%) COL1A1_01 Missing 4 0 Zero 145 (94.2%) 30 (20.7%) >0 9 (5.8%) 6 (66.7%) COL18A1_01 Missing 4 0 Zero 146 (94.8%) 32 (21.9%) >0 8 (5.2%) 4 (50.0%) CTGF_01 Missing 4 0 Zero 128 (83.1%) 25 (19.5%) >0 26 (16.9%) 11 (42.3%) LOX_01 Missing 4 0 Zero 149 (96.8%) 33 (22.1%) >0 5 (3.2%) 3 (60.0%) LOXL1_01 Missing 4 0 Zero 146 (94.8%) 31 (21.2%) >0 8 (5.2%) 5 (62.5%) LOXL2_01 Missing 4 0 Zero 115 (74.7%) 21 (18.3%) >0 39 (25.3%) 15 (38.5%) LOXL3_01 Missing 4 0 Zero 67 (43.5%) 5 (7.5%) >0 87 (56.5%) 31 (35.6%) LOXL4_01 Missing 4 0 Zero 122 (79.2%) 25 (20.5%) >0 32 (20.8%) 11 (34.4%) PLOD2_01 Missing 4 0 Zero 136 (88.3%) 32 (23.5%) >0 18 (11.7%) 4 (22.2%) PLOD1_01 Missing 4 0 Zero 111 (72.1%) 19 (17.1%) >0 43 (27.9%) 17 (39.5%) PCOLCE2_01 Missing 4 0 Zero 144 (93.5%) 32 (22.2%) >0 10 (6.5%) 4 (40.0%) PCOLCE_01 Missing 4 0 Zero 139 (90.3%) 31 (22.3%) >0 15 (9.7%) 5 (33.3%) PLOD3_01 Missing 4 0 Zero 109 (70.8%) 17 (15.6%) >0 45 (29.2%) 19 (42.2%) ITGB1_01 Missing 4 0 Zero 62 (40.3%) 8 (12.9%) >0 92 (59.7%) 28 (30.4%) FBLN2_01 Missing 4 0 Zero 139 (90.3%) 31 (22.3%) >0 15 (9.7%) 5 (33.3%) CYR61_01 Missing 4 0 Zero 50 (32.5%) 10 (20.0%) >0 104 (67.5%) 26 (25.0%) ITGA5_01 Missing 4 0 Zero 135 (87.7%) 26 (19.3%) >0 19 (12.3%) 10 (52.6%) ITGA3_01 Missing 4 0 Zero 56 (36.4%) 8 (14.3%) >0 98 (63.6%) 28 (28.6%) ITGA2_01 Missing 4 0 Zero 139 (90.3%) 29 (20.9%) >0 15 (9.7%) 7 (46.7%) ITGAV_01 Missing 4 0 Zero 120 (77.9%) 22 (18.3%) >0 34 (22.1%) 14 (41.2%) CSRC_01 Missing 4 0 Zero 90 (58.4%) 15 (16.7%) >0 64 (41.6%) 21 (32.8%) PTK2_01 Missing 4 0 Zero 61 (39.6%) 7 (11.5%) >0 93 (60.4%) 29 (31.2%) POSTN_01 Missing 4 0 Zero 103 (66.9%) 23 (22.3%) >0 51 (33.1%) 13 (25.5%) YAP_01 Missing 4 0 Zero 137 (89.0%) 26 (19.0%) >0 17 (11.0%) 10 (58.8%) CXCL1_01 Missing 4 0 Zero 94 (61.0%) 19 (20.2%) >0 60 (39.0%) 17 (28.3%) CSF2_01 Missing 4 0 Zero 131 (85.1%) 32 (24.4%) >0 23 (14.9%) 4 (17.4%) CCL2_01 Missing 4 0 Zero 112 (72.7%) 24 (21.4%) >0 42 (27.3%) 12 (28.6%) IL8_01 Missing 4 0 Zero 99 (64.3%) 15 (15.2%) >0 55 (35.7%) 21 (38.2%) IL6_01 Missing 4 0 Zero 62 (40.3%) 10 (16.1%) >0 92 (59.7%) 26 (28.3%) LAMA3_01 Missing 4 0 Zero 148 (96.1%) 34 (23.0%) >0 6 (3.9%) 2 (33.3%) TP53_01 Missing 4 0 Zero 125 (81.2%) 22 (17.6%) >0 29 (18.8%) 14 (48.3%) CDKN1A_01 Missing 4 0 Zero 118 (76.6%) 22 (18.6%) >0 36 (23.4%) 14 (38.9%) CDKN2A_01 Missing 4 0 Zero 103 (66.9%) 26 (25.2%) >0 51 (33.1%) 10 (19.6%) TAZ_01 Missing 4 0 Zero 133 (86.4%) 29 (21.8%) >0 21 (13.6%) 7 (33.3%) LAMC1_01 Missing 136 33 Zero 19 (86.4%) 0 (0.0%) >0 3 (13.6%) 3 (100.0%) LAMB1_01 Missing 4 0 Zero 109 (70.8%) 16 (14.7%) >0 45 (29.2%) 20 (44.4%) LAMA1_01 Missing 4 0 Zero 128 (83.1%) 30 (23.4%) >0 26 (16.9%) 6 (23.1%) LAMC2_01 Missing 5 0 Zero 145 (94.8%) 33 (22.8%) >0 8 (5.2%) 3 (37.5%) LAMB3_01 Missing 4 0 Zero 139 (90.3%) 33 (23.7%) >0 15 (9.7%) 3 (20.0%) GDF15_01 Missing 28 4 Zero 65 (50.0%) 10 (15.4%) >0 65 (50.0%) 22 (33.8%) DSPP_01 Missing 73 13 Zero 16 (18.8%) 7 (43.8%) >0 69 (81.2%) 16 (23.2%) NTRK3_01 Missing 28 4 Zero 130 (100.0%) 32 (24.6%)

[0078] To formulate a model that distinguishes melanoma that presents with regional metastasis at the time of diagnosis vs. no metastasis, logic regression was used. Logic regression is a machine learning technique that uses Boolean explanatory variables. There was not a typical technique to create good cut points for logic regression. To assign cut points in the variables, recursive partitioning followed by standardization of cut point levels was used. These were arbitrarily set at 0, 50, 250, and 500. Cut points derived by logic regression were adjusted to the next highest standard level. The cut point for ITGB3 was maintained at 0. The selected model for predicting metastasis was the following:

IF(OR(ITGB3>0,(AND(OR(PTK2>250,PLAT>500,PLOD3>250),CDKN2A<- 50)))=TRUE then predict metastasis Cut point ITGB3=0 Cut point PLAT=500 Cut point PTK2=250 Cut point PLOD3=250 Cut point CDKN2A=50

[0079] As can be seen from the formula, the risk of melanoma metastasis was high if ITGB3, PLAT, PTK2 or PLOD3 levels are increased and CDKN2A is low.

[0080] This model predicted regional metastasis (defined as a positive SLN biopsy at the time of primary cancer diagnosis) with a specificity of 80.3% and sensitivity of 97.3%.

OTHER EMBODIMENTS

[0081] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

444118DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 1gccaaccgcg agaagatg 18218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 2ggctggggtg ttgaaggt 18320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 3cgcgagaaga tgacccagat 20420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 4ggggtgttga aggtctcaaa 20521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 5tgacccagat catgtttgag a 21619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 6gtacatggct ggggtgttg 19718DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7ctgaacccca aggccaac 18820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8tgatctgggt catcttctcg 20920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 9aactctgcat tctcgcttcc 201020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 10gcagacagac actggcaaca 201121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 11gcaccattga aatcctgagt g 211220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12gctcccactt tgtctccagt 201322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13tcacagagga aactctgcat tc 221418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14ggacaccctc caggaagc 181518DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15atctccaggg gcaccatt 181621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 16agctgcacat cactcaggat t 211719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17actgctggcc acgagtacg 191820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18atgctccaca ggattcatgg 201920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19acagagctgt ggttggtgtg 202018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20ttgtcaattc ggccacct 182121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21tatctcctca gccaacagag c 212218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22agccaccaca ccaaccac 182319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23gtgtggccat gaatcctgt 192420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24ccacctccaa aaggatgctc 202520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25tctctctttc tggcctggag 202621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26gaatctttgg agtacgctgg a 212720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27tggaggctat ccagcgtact 202823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28cgtgagtaaa cctgaatctt tgg 232921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29ccagcgtact ccaaagattc a 213020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30tctctgctgg atgacgtgag 203120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31ggctatccag cgtactccaa 203221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32gctggatgac gtgagtaaac c 213320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33accattgagg acctgaggaa 203420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34gtccactgtg gctgtgagaa 203520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35cattgaggac ctgaggaaca 203620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36aatctgcaga aggacattgg 203720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37gatgacttcc gcaccaagta 203820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38cgcaggttca actctgtctc 203920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39tccgcaccaa gtatgagaca 204020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40actcatgcgc aggttcaact 204120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41gagcctcgtg actacagcaa 204222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42gcaggatgtt ggcattatca gt 224323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43aaaaccatcg atgaccttaa aaa 234420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44gatctgaagc aggatgttgg 204521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45ttcccaagtc aaatgatcca g 214620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46aagatggttc ccttgttcca 204720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47cggcatttgt tgctcagaat 204820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48gagcctgcat ttcaagttcc 204921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49ttccttcttc accatgcatt t 215020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50ggagccactg ctcaaaaata 205120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51tccaaagatc tgggctatga 205222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52ttgaaaagag tctgggtctg aa 225322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53gagaaaaact gtgaacctgt gg 225420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54ataagcaggt ggagcattgg 205522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55gaagacgaaa tggatacaga gc 225623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56gtgccaacat gaagactttt atc 235720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 57gtggtcagca cccagcttat 205819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 58ccaaggcctg cttcttgac 195920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59gctgtggtcc ttgcatctct 206020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60gcttcataag tctgcgccta 206119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61ctcctgcaca tgctttgga 196218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 62aggtctgcgg cagttgtc 186320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 63aggctttgga agtggtcatt 206420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 64ccattgtcat ggcaccatct 206524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 65gaagtggtca tttcagatgt gatt 246620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 66ccattgtcat ggcaccatct 206723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 67tggtcatttc agatgtgatt cat 236820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 68cattgtcatg gcaccatcta 206920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 69gtttcgcaga cctgacatcc 207020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70tcctcgtctg tagcatcagg 207120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71cctgacatcc agtaccctga 207220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 72tgaggtgatg tcctcgtctg 207320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 73gaatctccta gccccacaga 207421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 74ggtttcttca gaggacacag c 217522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 75cccatctcag aagcagaatc tc 227619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 76acagcattct gtggggcta 197719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 77ggaaaaccag gacccagag 197820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 78ctttttcccc tttgtcacca 207920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 79agaaaggtga acccggaaaa 208018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 80ggtttgcctc tgggtcct 188120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 81gagaaaaggg ccaaaaaggt 208220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 82catcccctga aatccaggtt 208320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 83aaagggccaa aaaggtgaac 208418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 84cctggcatcc cctgaaat 188520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 85gtgtcaacct gatggggaga 208620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 86gttaacgccc tgactgtggt 208720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 87ggtacagtgg gacagcaggt 208820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 88gatctgccat tgtggtaggc 208919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 89aaccacagtc agggcgtta 199018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 90gttcgtggcc cttccagt 189120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 91aagctgaagg tggaggggta 209220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 92gagtcacctg ctgtcccact 209320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 93tattcctccg aaccagcatc 209420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 94caccagctcc gagtcaatgt 209520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 95ccaccatcaa catggagaac 209622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 96agtcaatgtc cacagagaac ca 229720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 97caactgccac attggtgatg 209820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 98aaacctcctg ttggcctctt 209920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 99tgacatcacg gatgtgaagc 2010020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 100gggttgatga caacctggag 2010119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 101tgtgaccgag agcgagaag 1910221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 102caggctcagt tcaaagtggt t 2110320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 103cggacctttg tcgagtacct 2010420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 104gttgctctgc agtgccttct 2010520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 105gacttccgtt ggactgatgg 2010620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 106tggttgggtc tccaattctc 2010720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 107acgtgcaaga aaggaacagt 2010820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 108tccaaaggtc ttggcatttt 2010920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 109gcagagatgg agcactacgg 2011019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 110cagccttgaa tcctcatgc 1911119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 111ggaaggaatc gtggagcag 1911220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 112cagcagtggg aaccagtaca 2011322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 113ctgatgaatg aaatgaggag ga 2211420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 114cacaaatgag ccaaatccaa

2011521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 115cagtttgctg tgtgtttgct c 2111621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 116catgatttgg catttgcttt t 2111720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 117gccccaccag aggagtatgt 2011819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 118aagccgactt ccttcacca 1911920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 119gagaccattc ccctcctacc 2012021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 120gcttctgtgc catctcaatc t 2112120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 121cgaagctgac ctggaagaga 2012220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 122tgggagtacg gatgcacttt 2012318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 123gtgtgcaccg ccaaagat 1812419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 124cgtaccaccg aagatgcag 1912520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 125ctaccccggc tactacacca 2012620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 126gacaaaggcc aggtcaaact 2012719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 127agtcggggtg gattacgag 1912820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 128acagttgtag cgcaggaacc 2012919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 129atgatgatgg cagggatgg 1913020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 130gcattgatct cggcttcttg 2013120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 131ctgtggcaca caggaaacac 2013218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 132acgagggtca tgccacag 1813320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 133gccaaagacg ggtttcatta 2013420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 134gccatgattt tcttcccttc 2013519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 135ctcctcctac ggcaaggga 1913623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 136tggagattgt ctaaccagat ggg 2313720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 137ctcctacggc aagggagaag 2013821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 138ttgccagtac agtggagatt g 2113920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 139ccagagctgt gcagatgagt 2014023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 140tgcatctaga ttctttgcct ttt 2314119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 141agggcacagc agacttcct 1914220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 142tcgtccatgc tgtggtaatg 2014319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 143gcatgcacct ctcataccc 1914421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 144cgcattgtag gtgtcatagc a 2114519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 145cttggcagcc ttcctgatt 1914620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 146gcaaaactgc accttcacac 2014718DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 147cgctctgaag gggatctg 1814820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 148acagggtctg ccctctgact 2014920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 149gagctcagtc agagggcaga 2015021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 150aactttcccc gttttggtag a 2115121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 151tgaacagtgt ggatgagatg g 2115220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 152gcagggtgct ttggttgata 2015321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 153aagggtctcc agcacctcta c 2115420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 154aaggccttct catggatctt 2015519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 155gagctccgca aggatgact 1915619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 156aggacgaggg cgtagaggt 1915720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 157cattcaagga acccagaacc 2015820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 158gcgttgaaca aggtttcctc 2015920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 159aagagccaga gtgtcccaag 2016019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 160actgagagca ggaccacca 1916121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 161cttctcctgt gtccgctaca a 2116219DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 162catggcctga gcacatctc 1916321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 163tgcctgcacc tttaagaaag a 2116422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 164ccggtcaaac ttcttacact cc 2216519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 165aagggggaga tgtgctcag 1916618DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 166cagtccccac agctgcac 1816721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 167aaaccgaagt catagccaca c 2116819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 168aagctttccg cccattctt 1916920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 169aggcccaaga ctggctacat 2017020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 170ctgccatgac ctgttttcct 2017119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 171ggcaggtgcg aaccttatg 1917219DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 172ccttccagcc aatgttcct 1917319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 173gatcgctgag ctgaaggtg 1917418DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 174cggatgcccc atctgagt 1817521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 175cccattggcg agtttgagaa g 2117622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 176aggaagagtc gaaggtcttg tt 2217720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 177ggaagaaact gtggcagagg 2017820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 178ggacaggatt agctcccaca 2017920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 179acaacgttct gtcccccttg 2018020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 180ggggacagca tcaaatcatc 2018121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 181tggatgcaga tgttgttttg a 2118221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 182cacagctttc catgacgagt t 2118325DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 183ttgattgaac aaaacagaaa gatca 2518422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 184tgacgagtta caagaggagc aa 2218520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 185ctgctcatag cagccacctt 2018620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 186aggtgactgg ggcattgatt 2018720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 187acgtggagga ggacacagac 2018820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 188ggagccttca gggctacttc 2018919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 189agcactgcca aagtggatg 1919021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 190ttgttgacat ggaacaagac c 2119120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 191gtgggctaca tcagggtacg 2019220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 192cagagtcagc caccaactca 2019320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 193catcatctgg tccaacctca 2019420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 194gtcctcaggg atggtgtcat 2019520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 195tgacctcaag atgtgccact 2019620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 196tggttggggt caatccagta 2019722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 197gatggattcc agttcgagta tg 2219818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 198atcaggcgca ggaaggtc 1819919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 199cccaaaaaga gcgtcaggt 1920021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 200ttgttgacat ggaacaagac c 2120120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 201cttcctaaca gtccgcccta 2020220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 202cccgatcagc acagtgattt 2020320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 203ctgcttcagg gagacacacc 2020419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 204tggcttgcaa cttcctcac 1920520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 205aggaagttgc aagccaacaa 2020621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 206cgaccttccc ttaatcgtct t 2120719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 207gcggaggaaa actgtctgg 1920820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 208aaatctgagc agcaccctgt 2020920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 209atattcctgg gaatggcaca 2021023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 210ccatactgtg gtaatgttga tga 2321121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 211tgtcaacaac acagagggag a 2121220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 212cacgtagttg ctggggatgt 2021319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 213tggcaagatc accagacgg 1921420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 214ggcacctttc gtggtctcac 2021520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 215catgtcgtct tggctcactc 2021621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 216aaattctggc cccaacaata c 2121720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 217catgtcgtct tggctcactc 2021821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 218aaattctggc cccaacaata c 2121918DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 219aggagccagc gtctaggg 1822019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 220ctgcccatca tcatgacct 1922120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 221aacgcaccga atagttacgg 2022220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 222catcatcatg acctggatcg 2022320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 223cactgttacg attcccctga 2022420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 224cggctttctc atcaggtttc 2022520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 225attagacggc ctcctgcatc 2022620DNAArtificial SequenceDescription of Artificial

Sequence Synthetic primer 226agaccagccc ctcttcatct 2022719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 227ggccccggga attatatct 1922820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 228ccacttcata gtgggggttc 2022919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 229ctgcacaact gccacacag 1923020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 230gttctgcatt ggctgggtat 2023119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 231cgtggcaagt gaggggttc 1923221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 232cgaagactcg gaatgagagg g 2123318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 233gaggcttcct gctctggt 1823419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 234cgcaaaattg gtgctcagt 1923520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 235gtccgggact tcctaacaga 2023620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 236gctgacctcc tggatagtgg 2023720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 237gtggtcagca cccagcttat 2023819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 238ccaaggcctg cttcttgac 1923920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 239gctgtggtcc ttgcatctct 2024020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 240gcttcataag tctgcgccta 2024120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 241cttccctcag ctttcaggac 2024220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 242tctggggtcc tagggaattg 2024320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 243acctcaagat gtccctcagc 2024419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 244caggagggtc ctgtacgtg 1924520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 245gagaccttcg gcgagtacag 2024620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 246aaaggccttc tcctcgttgt 2024720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 247ggcggcaaat actcagtgaa 2024820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 248cctgggtgtc ctccttatcc 2024920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 249cggatactca cgccagaagt 2025020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 250agagatacgc aggtgcaggt 2025120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 251aagattcgaa caccgacctc 2025220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 252gcacttctgg cgtgagtatc 2025318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 253cacgcctgga acaaggac 1825420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 254atgcacctca tggttgttgg 2025519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 255caggtgacag gcatcgact 1925620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 256cgcaggtcac aatacggtta 2025720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 257tgaggccctg agagagaaga 2025820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 258attccgaaac tccacacgtc 2025920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 259tcaaggaaat tgagcaacga 2026020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 260tctgatttgg gcatttgagc 2026119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 261tatggtcgac ggtccaaat 1926220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 262tcctcaccac tgatgacagc 2026320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 263cactgtgacc cacaaaccag 2026420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 264gcaagtccaa ctgctatgga 2026520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 265tgaggccctg agagagaaga 2026620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 266attccgaaac tccacacgtc 2026720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 267tctactcctt tgggcgagtg 2026820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 268cgtccttcag gggaattctt 2026920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 269taaggacacc ccaaattcca 2027020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 270gccaagatga tcagccattc 2027121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 271gcagtcaaca gtcgaagaag g 2127220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 272agctttttct tctgcccaca 2027320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 273agccagcaag cagtgaagtc 2027420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 274tcaggtgact caagcagtgg 2027520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 275ccgggagtct atggtcaaac 2027620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 276cacggcactc agcttacttg 2027719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 277tggagcagtc ttcgtttcg 1927820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 278ctggctcctc ttctgaatcg 2027919DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 279gcccgattca gaagaggag 1928021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 280tcatctctgc agatcacttg g 2128120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 281ccagcagagg cataaggttc 2028220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 282agtagtgcct tcgggactgg 2028324DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 283aggctgattc tggaagttct gagg 2428421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 284aatctggact gcttgtggct g 2128519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 285gttgggcctc caggattta 1928620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 286gcctggtagt cctgggaaac 2028720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 287tggatggatt gtgttcctga 2028820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 288gcctgccttc aagatttctg 2028921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 289ctgagactgt gctgacctgt g 2129020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 290ctcttcatct ccgccttctg 2029120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 291gagaccgcct acatcgaaga 2029220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 292ggtagcgttc aaacctcctg 2029320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 293acaccgtcct caacctgaag 2029420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 294aatggccagt gccacatagt 2029520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 295ggtgcacttt gtgagcaaga 2029620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 296ttggtatgca gatgggttca 2029720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 297agctgtggtc caacttctgg 2029820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 298gtgtggtaac cgggaaacag 2029921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 299ttcagtttgc tgtgtgtttg c 2130020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 300ccaccttctg gagaatccaa 2030120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 301ggcagtattg acagggagga 2030220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 302tactcttgct ggaggctggt 2030322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 303gcctattctg tcacttcggc tc 2230422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 304gcaggcacag gtcttgatga ac 2230520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 305gacctctggg aggtgttcag 2030620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 306ttagggatcg acgaaggaga 2030720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 307attcctgcca tcaaccagac 2030820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 308cctgcttctt ggcttcattc 2030920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 309caaagggaca tcccaaaatg 2031022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 310gagtcagcca tgattttctt cc 2231120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 311taccccgagt acttccagca 2031220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 312gatctgcttc caggtcttgc 2031320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 313cacacagaca gccactcacc 2031420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 314caggggtggt tattgcatct 2031520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 315cagaccccaa ctatgtgcaa 2031621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 316cgcattgtag gtgtcatagc a 2131719DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 317ctctcttggc agccttcct 1931821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 318tgaattctca gccctcttca a 2131920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 319tcgccttagt cgtcaccctt 2032023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 320tgtttctcgt caactccacc tcg 2332120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 321ctgatttcat cggggttgtc 2032220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 322tgccttgctg aatgaacttg 2032319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 323ccagtgaaac agccaccac 1932420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 324ctccttccag tgttccaagg 2032520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 325ggactctgtc acacccacct 2032620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 326cagggtctca gggaggtctt 2032721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 327tgccagagct gagatgttgt t 2132820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 328tgtagcattt cggctttcct 2032920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 329ggcaagtact gcgagtgtga 2033020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 330attcttttcg gtcgtggatg 2033119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 331cactgctgct cctgctcct 1933221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 332tgttcagcat cttttcgatg a 2133321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 333tgacaatgac aacatcccag a 2133420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 334tgagtctgcc atgacctgtt 2033521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 335ccctgctcta cacagaacca g 2133619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 336acacctggct cccctttct 1933724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 337gcctggatct tctttctcct ttgc

2433822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 338catccagggc gatgtacttg tc 2233920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 339ccccctctga gtcaggaaac 2034020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 340tcatgtgctg tgactgcttg 2034122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 341tggacccacc aagattctcc tg 2234222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 342gaccacagct ttccatgacg ag 2234320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 343tctgtgcctg ctgctcatag 2034420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 344gagtttgggt ttgcttgtcc 2034518DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 345cgagaagtgc ccaggaag 1834620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 346agtgagaagc caggaaagca 2034720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 347tggaaatatc acccacagca 2034820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 348aggcattttt gcttcacacc 2034920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 349gctccagctt ctacgtggtc 2035021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 350ttaattatcg aagcggtcga a 2135120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 351agccagcaga tcgagaacat 2035220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 352ccttcttgag gttgccagtc 2035320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 353caccaatcac cccattaacc 2035420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 354gcttgagctg agctttttcc 2035520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 355ccaggtgctg gaaaaagaag 2035620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 356gagctgctct gcctgagtct 2035720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 357gcagacacac ctgttggaaa 2035820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 358gaacgacctt cccttaatcg 2035922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 359cctactacat ccaggcgtcc ac 2236021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 360atgcaaatcg cctgtggtag c 2136120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 361ctgttcggag gcttcaactc 2036218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 362agggatctcc caggcatc 1836320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 363tacctgggat cacctccatc 2036420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 364acagggatcc tcagtgtcgt 2036519DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 365cgggatgagt tgggaggag 1936620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 366ttagggcttc ctcttggaga 2036718DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 367atggtgcgca ggttcttg 1836818DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 368accagcgtgt ccaggaag 1836918DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 369gagcagcatg gagccttc 1837020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 370gcatggttac tgcctctggt 2037120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 371caaacagaca aggctggtga 2037222DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 372tcaatctcat ctggattttt gg 2237320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 373ctgcaggtgg acaacagaaa 2037420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 374catcagccag aatcccatct 2037520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 375gtccccagag agacctgttt 2037619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 376agacacaatt ggcgcaggt 1937720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 377aagactggac gcgatagctg 2037820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 378ggttgttcct gagacgctgt 2037920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 379tacaccagac ccgtgttcct 2038022DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 380tccaggtcaa acttctcgaa gg 2238118DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 381cttcaatgcc cagctcca 1838220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 382ttcccaacca catcttccac 2038318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 383ctgctgctct tgggcact 1838420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 384cagcagtcaa aggggatgac 2038521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 385aggattccta tgtgggcgac g 2138621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 386tcaggcagct cgtagctctt c 2138723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 387ggaatgtggg ctttgtgttc acc 2338821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 388aggccaggac tcgtttgtac c 2138921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 389acatcaaggg catcgtcaag g 2139024DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 390tctctttctc ctgcacagtc ttgg 2439122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 391tgctcgcgct actctctctt tc 2239220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 392tcacatggtt cacacggcag 2039320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 393tggccttctc tctggaaatg 2039420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 394tcatttcctc ctcgtggttc 2039520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 395aggtgaccat gcagaacctc 2039620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 396cctcgtggtt cttcttcagg 2039720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 397gaaatcttgg gcttgatgga 2039820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 398ccgaggttgt tgttgaaggt 2039920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 399ccatggataa agctgccaat 2040020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 400gacacagcaa gctcacaagc 2040120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 401cactcttaca ccacggctga 2040220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 402cataagcagg tggagcattg 2040322DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 403ttgtccaggg tattgaaagt gc 2240421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 404gacaagagca gaagatgcgg g 2140521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 405gcgttggaac agaggttgga g 2140622DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 406caggtgtctc aagggtagca gg 2240721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 407cttccctttc gccacagtat g 2140822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 408cctccttctc cttcttgcca ct 2240922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 409aatggctctc agatgctcct gg 2241021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 410gattctgcca gcagttggtc c 2141120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 411accacagctt cctggtggag 2041221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 412gagcggatgg gcttcttgat g 2141321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 413aacgtgacct tgtctcccaa c 2141420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 414gcatgacatc gccgattctt 2041521DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 415ttcaacaagc ccacccacta c 2141621DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 416gttctcaatg acagggatgc g 2141722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 417catggaatac ctggagggca ac 2241821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 418caggtgccag cagttcttca t 2141922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 419tctcagagct tctctacatc ac 2242021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 420ctgacgactc cttgttcacc a 2142121DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 421tcacctcctc tgtaccattg c 2142220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 422ctcatctcca atgccctcga 2042321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 423tgcaatgaag agagggctct g 2142422DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 424cgtggccctg gtatctattt ca 2242520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 425ggctacagct tcaccaccac 2042620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 426taatgtcacg cacgatttcc 2042718DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 427cccagccagg aaatccat 1842820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 428ctggctcctc ttctgaatcg 2042920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 429cagtgcctgt caaaagttgc 2043018DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 430ccccgttgaa acaccttg 1843120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 431gaccttggaa acccaatgaa 2043220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 432tccatctctg actgctggtg 2043320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 433tgctatccct gtacgcctct 2043420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 434gagtccatca cgatgccagt 2043520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 435ggacttcgag caagagatgg 2043619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 436cttctccagg gaggagctg 1943720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 437gatgcagatt gagagcctga 2043820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 438ttcttcaggt aggccagctc 2043921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 439ggtggtatgt gaccttggaa a 2144020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 440gcacactgaa acgaagacca 2044120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 441gaaggatttg ctgggaagtg 2044220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 442cgtggccctg gtatctattt 2044323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 443tacttggaaa atcacagttc ccg 2344423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 444tgaatcggaa attgagaacg act 23

Claims

1. A method for identifying a malignant skin lesion, wherein said method comprises: (a) determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain a measured expression level of said marker gene for said test sample, (b) determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample, (c) removing, from said measured expression level of said marker gene for said test sample, a level of expression attributable to keratinocytes present in said test sample using said measured expression level of said keratinocyte marker gene for said test sample and a keratinocyte correction factor to obtain a corrected value of marker gene expression for said test sample, and (d) identifying said test sample as containing a malignant skin lesion based, at least in part, on said corrected value of marker gene expression for said test sample.

2. The method of claim 1, wherein said keratinocyte marker gene is K14.

3. The method of claim 1, wherein said marker gene is SPP1 or ITGB3.

4. The method of claim 1, wherein step (c) comprises (i) multiplying said measured expression level of said keratinocyte marker gene for said test sample by said keratinocyte correction factor to obtain a correction value and (ii) subtracting said correction value from said measured expression level of said marker gene for said test sample to obtain said corrected value of marker gene expression for said test sample.

5. The method of claim 1, wherein said marker gene selected from the group consisting of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1.

6. The method of claim 1, wherein said marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC.

7-8. (canceled)

9. The method of claim 1, wherein said method comprises determining, within said test sample, the expression level of at least seven marker genes selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain measured expression levels of said at least five marker genes for said test sample.

10. The method of claim 1, wherein said method comprises determining, within said test sample, the expression level of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1.

11. The method of claim 1, wherein said method comprises determining, within said test sample, the expression level of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC.

12. A kit for identifying a malignant skin lesion, wherein said kit comprises: (a) a primer pair for determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain a measured expression level of said marker gene for said test sample, and (b) a primer pair for determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample.

13. The kit of claim 12, wherein said keratinocyte marker gene is K14.

14. The kit of claim 12, wherein said marker gene is SPP1 or ITGB3.

15. The kit of claim 12, wherein said marker gene selected from the group consisting of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1.

16. The kit of claim 12, wherein said marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC.

17-18. (canceled)

19. The kit of claim 12, wherein said kit comprises primer pairs for determining, within said test sample, the expression level of at least seven marker genes selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, CDKN2A, and CXCL1 to obtain measured expression levels of said at least five marker genes for said test sample.

20. The kit of claim 12, wherein said kit comprises primer pairs for determining, within said test sample, the expression level of ITGB3, PLAT, PTK2, PLOD3, CDKN2A, SPP1, TNC and COL4A1.

21. The kit of claim 12, wherein said kit comprises primer pairs for determining, within said test sample, the expression level of PLAT, SPP1, TNC, ITGB3, COL4A1, PTK2, PLOD3, and SPARC.

22. A method for identifying a malignant skin lesion, wherein said method comprises: (a) determining, within a test sample, the expression level of a marker gene selected from the group consisting of PLAT, SPP1, TNC, ITGB3, COL4A1, CD44, CSK, THBS1, CTGF, VCAN, FARP1, GDF15, ITGB1, PTK2, PLOD3, ITGA3, IL8, and CXCL1 to obtain a measured expression level of said marker gene for said test sample, (b) determining, within said test sample, the expression level of a keratinocyte marker gene to obtain a measured expression level of said keratinocyte marker gene for said test sample, (c) removing, from said measured expression level of said marker gene for said test sample, a level of expression attributable to keratinocytes present in said test sample using said measured expression level of said keratinocyte marker gene for said test sample and a keratinocyte correction factor to obtain a corrected value of marker gene expression for said test sample, and (d) identifying said test sample as containing a malignant skin lesion based, at least in part, on said corrected value of marker gene expression for said test sample.

23. The method of claim 22, wherein said keratinocyte marker gene is K14.

24. The method of claim 22, wherein said marker gene is SPP1.

25. The method of claim 22, wherein step (c) comprises (i) multiplying said measured expression level of said keratinocyte marker gene for said test sample by said keratinocyte correction factor to obtain a correction value and (ii) subtracting said correction value from said measured expression level of said marker gene for said test sample to obtain said corrected value of marker gene expression for said test sample.